Coexistence of chronic hyperalgesia and multilevel neuroinflammatory responses after experimental SCI: a systematic approach to profiling neuropathic pain

Background People with spinal cord injury (SCI) frequently develop neuropathic pain (NP) that worsens disability and diminishes rehabilitation efficacy. Chronic NP is presently incurable due to poor understanding of underlying mechanisms. We hypothesized that multilocus neuroinflammation (NIF) might be a driver of SCI NP, and tested it by investigating whether NP coexisted with central NIF, neurotransmission (NTM), neuromodulation (NML) and neuroplasticity (NPL) changes post-SCI. Methods Female Sprague–Dawley rats (230–250 g) with T10 compression or laminectomy were evaluated for physical conditions, coordinated hindlimb functions, neurological reflexes, and mechanical/thermal sensitivity thresholds at 1 day post-injury (p.i.) and weekly thereafter. Eight weeks p.i., central nervous system tissues were histochemically and immunohistochemically characterized for parameters/markers of histopathology and NIF/NTM/NML/NPL. Also analyzed was the correlative relationship between levels of selected biomarkers and thermosensitivity thresholds via statistical linear regression. Results SCI impaired sensorimotor functions, altered reflexes, and produced spontaneous pain signs and hypersensitivity to evoked nociceptive, mechanical, and thermal inputs. Only injured spinal cords exhibited neural lesion, microglia/astrocyte activation, and abnormal expression of proinflammatory cytokines, as well as NIF/NTM/NML/NPL markers. Brains of SCI animals displayed similar pathophysiological signs in the gracile and parabrachial nuclei (GrN and PBN: sensory relay), raphe magnus nucleus and periaqueduct gray (RMN and PAG: pain modulation), basolateral amygdala (BLA: emotional-affective dimension of pain), and hippocampus (HPC: memory/mood/neurogenesis). SCI augmented sensory NTM/NPL (GrN and PBN); increased GAD67 (PAG) level; reduced serotonin (RMN) and fear-off neuronal NTR2 (BLA) expressions; and perturbed neurogenesis (HPC). Conclusion T10 compression caused chronic hyperalgesia that coexisted with NIF/NTM/NML/NPL responses at multilevel neuroaxis centers. The data have provided multidimensional biomarkers as new mechanistic leads to profile SCI NP for therapeutic/therapy development. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-022-02628-2.

Introduction
Traumatic spinal cord injury (SCI), either moderate or severe, can produce long-term sensorimotor deficit, autonomic abnormality, respiratory disorder, lower urinary tract dysfunction, and additional debilitating sequelae including neuropathic pain (NP) and mood disorder [1,2]. It has been reported that ≥ 80% of all individuals surviving acute SCI experience severe pain, and 40-60% of them develop chronic pain [3]. Notably, chronic NP, which remains incurable, has been diagnosed in ≥ 53% of people with SCI, jeopardizing life quality and rehabilitation opportunity, and imposing socioeconomic burden [1][2][3]. Effective clinical management for SCI NP and control of its biological and/or psychological triggers are unmet medical needs [4][5][6]. In therapeutic development, laboratory interventions have aimed to suppress specific neural facilitation of pain [7], activate inhibitory interneurons and neuromodulation (NML) [8,9], reduce pain neurotransmission (NTM) [10,11], or impede neuroinflammation (NIF) [12]. Therefore, the broad mechanistic diversity of post-injury (p.i.) NP has likely played a major role in preventing conventional therapeutic tactics from exerting long-term efficacy and avoiding serious side effects [13][14][15].
An earlier study of ours demonstrated that administration of the multimodal effecting drug huperzine A, which concurrently stimulates inhibitory interneurons and suppresses NIF via elevating cholinergic tone, and mitigates excitotoxicity and sensory neuronal activity by blocking NMDA receptors, ameliorated NP in rats with T10 compression [14]. Clinically, neuroimaging uncovered that SCI NP increased intraspinal cord NIF metabolite levels [15]. These data suggested that local and systemic inflammation may act as pathogenic factors to induce the evolvement of NP, pain-triggered complications, and other neural disorders p.i. (e.g., affective components of pain, distal motor neuron/neuromuscular junction abnormalities, and axon demyelination) [15][16][17][18][19][20][21][22]. However, to date, little has been done to systematically investigate NIF in the central nervous system (CNS) using clinically relevant SCI models. Here, we hypothesized that NIF in central sensory relay and pain modulation nuclei might be a primary driver of chronic SCI NP, and tested the hypothesis by first probing whether T10 compression caused long-term coexistence of NP and multilocus NIF responses in female rats. Since human NP is difficult to model [23], we utilized physical signs of pain, evoked hypersensitivities, plus sensorimotor and NIF/ NTM/NML/NPL outcome measures to generate multidimensional biomarkers as novel mechanistic leads to systematically profile SCI NP, aiming to improve the study's clinical relevance [24].

Animals, SCI modeling, and perioperative managements
Young adult female Sprague-Dawley (SD) rats (230-250 g; n = 24) were purchased from Charles River Laboratories (Wilmington, MA), and housed in pairs at the Animal Research Center of the Boston Children's Hospital under a 12-h light/dark photocycle in a colony under ambient temperature and humidity with food and water available ad libitum [14,25,26]. T10 compression was performed according to a standardized protocol [14,26]. Concisely, rats were anesthetized with ketamine (75 mg/ kg, i.p.)/xylazine (10 mg/kg, i.p.) and placed on a heating pad (37 °C) that was irrigated with an EZ-200 water circulation pump (E-Z Systems Inc., Palmer, PA, USA). Buprenorphine was administered (0.05 mg/kg, s.c.) for pre-emptive analgesia. A midline incision, soft tissue dissection, and laminectomy were made at the 10th thoracic vertebral level (T10: corresponding to T11 spinal cord). Hemostasis was induced with normal saline and Gelfoam ® (Pfizer). After the body trunk was suspended (~ 1 cm clearance) with a Rodent Spine Stabilization Frame custom-made by the Scientific Instrument Shop, Harvard University School of Engineering and Applied Sciences (Cambridge, MA) [26], a 35 g stainless steel impounder was lowered gently upon the dura via a micromanipulator (MM 33, Märzhäuser) to deliver a 5-min standing load of the weight (i.e., a moderate compression regimen) [26]. For rats that received T10 laminectomy only as sham surgery controls, no weight loading was performed. The muscles were then sutured (3-0 Ethilon black brand, Ethicon ® ), and skin stapled with wound clips (Reflex ® ; Cellpoint Scientific, Gaithersburg, MD) [14,26].
Following the animal protocol, signs of stress and pain, which included reduced grooming, porphyrin staining around the eyes, vocalization, etc., were routinely monitored and analyzed based on a formula we developed to determine the likelihood of spontaneous pain presence (Additional file 1: Table S1).

Experimental design
The experiments were conducted largely following a randomized block design. The sample size was determined on the basis of a power analysis from a previous study on the same SCI model [14]. With the α value (probability of rejecting H 0 when H 0 is true) being set at 5%, an average sample value of 0.41 g (post-SCI baseline sensory threshold), mean test value (post-treatment) of 3.1 g, and group size of four rats, the statistical power (i.e., 1-β) equaled ∼ 100%, indicating that the β value (i.e., the probability of not rejecting H 0 when H 0 is false) equaled ∼ 0%. All animals were habituated for ≥ 3 days, during which the baseline data were collected, which included body weight and outcomes of modified von Frey filament (mVF), locomotion, inclined plane, spinal reflex, and hot plate tests (Fig. 1A). All qualified rats received 3 rounds of functional magnetic resonance imaging [fMRI] examinations (pre-surgery, and 2 and 8 weeks after operation) for a separate study conducted by investigators of an independent laboratory.
The SCI and laminectomy control groups (n = 14 and 10, respectively) were behaviorally tested at 1 day and weekly thereafter for 8 weeks p.i. (Fig. 1A, B). Behavioral Fig. 1 The experimental design, SCI modeling, and coordinated hindlimb functional changes. A The time course of major experimental procedures comprised a brief habituation, T10 compression and postsurgical care, and behavioral evaluations performed weekly for 8 weeks. B Left: this schematic diagram illustrated the surgery setting of T10 moderate quasi-static compression (35 g × 5 min). Middle: a zoomed in presentation of the compression injury epicenter. Right: the contents of the performed behavioral tests. C The day 1 and weekly post-injury (p.i.) group average BBB locomotor scores of the SCI rats (red) versus those of the laminectomy control animals (blue) showed significant locomotion deficits resulting from SCI. D Left: the inclined plane test was done with rats facing downward. Right: compared to the laminectomy sham surgery, T10 compression resulted in significantly reduced group mean maximum angle where the animals could hold their position steady for 5 s (for both BBB and incline plan tests: *p < 0.05 to < 0.001; two-way repeated measures ANOVA with Sidak's post hoc test; n = 12/SCI group; n = 10/laminectomy control group) evaluation was performed during the 12-h light photocycle, and mostly by two examiners to obtain mutually agreed scores following the same test order (i.e., mVF → open-field locomotion → incline plate → proprioceptive positioning/placing reflex → other neurological reflexes → ~ 30-min break → bilateral hot plate → ~ 30-min break → unilateral hot plate; note: per convention, motor and sensory data were separately presented). At the end of the study (≥ day 56 p.i.), animals were euthanatized, and the spinal cord and brain tissues were fixed by intracardiac perfusion. All animals except two were investigated over the entire study duration (n = 22: 12/SCI and 10/laminectomy). For the two animals excluded, one was inadequately injured (i.e., no loss of micturition reflex), and the other was euthanatized due to a skin lesion (note: its data were not included). Data values were expressed as mean ± SEM. Statistical tests were conducted with significance level set at p < 0.05. The experimental procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals (US National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 8th edition) after review and approval by the Institutional Animal Care and Use Committee (IACUC) of Boston Children's Hospital and the US Department of Defense.

Evaluation of coordinated hindlimb functions Locomotion
The hindlimb locomotion was assessed with the Basso, Beattie and Bresnahan (BBB) locomotor rating scale following the original protocol [28]. Briefly, animals were individually placed in an open, flat area staged by a rubber pad with a patterned surface texture (Fig. 1C). Bilateral hindlimb locomotor function was scored according to a 21-point scale (i.e., 0 for complete paralysis and 21 indicating normal function). The score comprehensively demonstrated hindlimb open-field locomotor aptitude.

Incline plane test
This assay quantifies a rat's ability to maintain coordinated body posture control on an inclined plane (Fig. 1D) [25][26][27]. Specifically, animals were individually positioned facing downward on a 40 × 30 cm plastic board covered with a rubber mat that had a fine groove surface pattern. The steepest degree of inclination at which a rat could hold its body posture for 5 s on two consecutive trials when placed vertically on the board and facing downward was recorded [25][26][27].

mVF test (mVFT) of mechanical hyperalgesia and tactile allodynia
Rats were placed on a metal mesh floor inside a Plexiglas box. After ~ 3 min of acclimation, a series of von Frey hairs, which are nylon monofilaments of 50 mm in length and in varied diameters/stiffnesses (i.e., logarithmically incremental stiffness scaling from 0.008 to 300 g) exerting different forces (typical force range for the presented study: 0.04-60 g), were applied to the plantar surface of the forepaw and hindpaw, and dorsal skin of the T11-T12 dermatomes to detect above-, below-, and at-injury level hypersensitivity, respectively ( Fig. 2F-I) [14]. The filament was applied with sufficient force to cause the fiber to bend to produce a punctate stimulus to a given area of the body for a total of ~ 5 s. Recorded as positive reactions were brisk withdrawal, paw licking, or body flinching that were evoked by the minimum threshold (unit: g).

Hot plate test to measure thermal hypersensitivity
The hot plate test utilizes latency measurements to evaluate acute, cutaneous hypersensitivity involving the supraspinal neural network response in rodents to noxious thermal stimuli [29]. In this study, bilateral and unilateral hot plate tests were done following published protocols [30,31]. The metal plate surface of the apparatus (IITC Life Science, Woodland Hills, CA) was maintained at 52 °C. In the bilateral test (Fig. 2J), rats were individually habituated on a non-heated (i.e., room temperature) metal surface for 5 min before each was placed on the pre-heated plate with its locomotion territory restricted by a Plexiglas arena. The temporal latency to hindpaw licking and quick shaking, or jumping, was recorded, at which point the rat was swiftly removed. In the unilateral test (Fig. 2K), each rat was gently restrained to allow the plantar side of the tested hindpaw to contact the hot plate (52 °C) to measure the withdrawal latency of each paw separately, with the shorter one recorded as the animal's thermosensitivity threshold. Three tests for either side were conducted separately and alternately at 2-min intervals in order to obtain the mean latency time as results. To prevent any occurrence of excessive thermal pain or injuries, the maximum time for each round of heat exposure was restricted to 30 s for both test types [30,31].

Tissue preparation
After all behavioral tests were completed 8 weeks p.i., animals were euthanatized with i.p. injection of 90 mg/ kg ketamine and 15 mg/kg xylazine. Tissue was fixed via intracardiac perfusion with phosphate buffer (PB; pH 7.4) Fig. 2 The data of sensorimotor behavioral evaluations of neurological reflexes, mechanical hypersensitivity, and bilateral or unilateral thermal hypersensitivity. A-E Spinal neurological reflexes. At day 1 post-injury (p.i.), SCI rats showed severe loss of contact righting reflex (A), proprioceptive positioning reflex of paw placing (B), and withdrawal reflex in response to brief extension of joints (C), which recovered overtime, with 80% SCI rats performing normally by 3 (A, C) or 4 weeks p.i. (B). Withdrawal reflexes caused by pressuring (D) and brief pinching (E) of the hindpaw exhibited discernible abnormalities p.i., with their recovery rates noticeably slower and more incomplete, relative to that of the righting (A), placing (B), or extension (C) reflex. By 8 weeks p.i., only ~ 33% SCI rats showed a normal nociception reflex (E). F-H Results of the modified von Frey filament tests of mechanical hypersensitivity (mVFT; F). In contrast to a stable array of sensitivity thresholds of the control group, at day 1 p.i., the mean threshold to trigger a withdrawal response following mVF stimulation drastically increased in the forepaws (G; 11.1 ± 1.5 g) and hindpaws (H; 45.8 ± 5.1 g) in the SCI rats. Afterward, the group mean threshold, compared to the pre-SCI level and control group, significantly decreased in the forepaws (i.e., above-injury level allodynia; G) and hindpaws (i.e., below-injury level allodynia; H), starting in 2 weeks after SCI. At-injury level allodynia measured in the dorsal dermatomes adjacent to the T10 injury had a comparable pattern of hypersensitivity (I; *p < 0.01 to < 0.001; n = 12/SCI or 10/control; two-way repeated measures ANOVA with Sidak's post hoc test). J, K Data of bilateral and unilateral hot plate tests (i.e., thermal hypersensitivity/ allodynia). In the bilateral hot plate test, the pre-SCI and pre-laminectomy mean hindlimb latencies were statistically indistinguishable and the control group showed steady postsurgical response latencies (J). At day 1 p.i., the group mean latency markedly increased (19.1 ± 3.1 s) in the injured rats. Starting in week 3 after SCI, thermal hypersensitivity developed, which remained continuous and significantly different than the baseline values and those of the control group (J; SCI: n = 12, laminectomy: n = 10; *p < 0.05-0.001; two-way repeated measures ANOVA with Sidak's post hoc test). The unilateral hindlimb hot plate test revealed a similar pattern of response latency changes. However, this test detected significantly reduced group mean latencies 1 week earlier, starting on day 14 p.i., compared to the baseline level and control group (K; n = 12/SCI or 10/control; *p < 0.05 to < 0.001; two-way repeated measures ANOVA with Sidak's post hoc test; note: to make the figure succinct, the gentle body holding logistics were not depicted in the inset) and 4% paraformaldehyde (PFA; pH 7.4). Afterward, spinal cords and brains were collected for overnight postfixation in 4% PFA, followed by sequential dehydrations in 10-30% sucrose solutions before freezing in 2-methylbutane/isopentane (Sigma-Aldrich, St. Louis, MO) at − 50 °C for cryostorage (− 80 °C). For microscopic morphometric and immunohistochemical (IHC) analyses, the tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA). Spinal cord cryosectioning generated circulation series of 20-μm tissue slices of paired injured and laminectomy spinal cords (5 section pairs/per slide × 10; frosted glass slides; Thomas Scientific, Swedesboro, NJ). The arrangement produced sections that comprised ∼ 1000 μm thickness of the spinal cord in each series of 10 slides [25][26][27]. This formula was adapted for brain sectioning: each series consisted of 5 slides with each having 3 × 40-μm-thick sections (i.e., 25 sections per series of ∼ 1000 μm thickness of brain tissue; tissue collection rate: 60%).

Histopathological analysis
For morphological analysis, every 10th (spinal cord) and 5th (brain) tissue section slides of each animal were stained with solvent blue (Sigma-Aldrich, St. Louis, MO) plus hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO) as per our standard protocols [26,32]. The stained tissue sections were imaged on an Axiovert 200 microscope with a digital Axiocam camera (Carl-Zeiss Microimaging). Image analysis was conducted using ImageJ ® (National Institutes of Health) and Photoshop ® (Adobe, San Jose, CA) to identify the lesion epicenter and quantify gray matter and white matter sparing as per methods previously published [27].
Per established protocols [14,22], the paired sections were quickly washed, hydrated, and perforated using phosphate-buffered saline (PBS) with 0.03% Triton X-100 (PBST; Sigma-Aldrich) before blocking non-specific binding sites with 4% (vol/vol) normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBST for 1 h at room temperature. Different primary antibody solutions were added for incubations at 4 °C overnight, followed by quick PBST washing and reactions (1 h) with the corresponding secondary antibodies (Table 1) at room temperature. After washing, the slides were cover-slipped with Vectorshield ® Anti-Fade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA) for evaluations (see Additional file 2: Table S2 for IHC protocol specifics). In addition, tissue sections of both experimental groups for staining of molecular markers CGRP, p75NTR, serotonin (5HT), and Homer-1a were first processed with antigen retrieval treatment (Additional file 3: Table S3). Briefly, the slides were processed according to Steps 1-5 as described in Additional file 2: Table S2 before incubation at 85 °C for 15 min with tissue sections immersed in the antigen retrieval buffer (i.e., 1× citrate-based antigen unmasking solution; Vector Laboratories, Burlingame, CA). After PBS washing, the slides were subjected to procedures detailed in Steps 6-16 in Additional file 2: Table S2 (see  Additional file 3: Table S3 for more details).
For qualitative assessment and semi-quantification of specific IHC signals, confocal imaging was conducted through a Nikon C2 Laser Scanning Confocal Microscope equipped with NIS-Elements Software 4.30.1 (Nikon, Melville, NY). To determine IHC reaction specificity, the orthogonal slices were collected to reconstruct z-stack 3D images of 10-15 μm thickness consisting of 1-μm steps by using NIS-Elements (Nikon). The positive signal threshold range of each marker was attained by averaging the pixel brightness of the weakest positive labeling signals and that of strongest signals against the average background luminance level [14,26]. Counting of positive IHC pixels for each marker was performed with ImageJ ® (NIH) and Photoshop ® (Adobe) to compute the percentage of the area that contained qualified signals of each antigen [i.e., immunoreactivity level (IRL)], relative to the entire visual field comparably selected for all samples. Finally, IRLs of the same IHC signals in the immediately adjacent confocal images were also computed to ensure the specificity and consistency of the IHC outcomes.

Statistical analysis
Statistical computations were done using GraphPad Prism (version 7.0, GraphPad Software, San Diego, CA). Unless otherwise specified, the data of locomotion, incline plane, mVFT, hot plate tests, and histopathology were evaluated by two-way repeated measures ANOVA with Sidak's post hoc test suggested by the software based on data features. To compare signal area scales (i.e., IRL) of IHC markers between the SCI and laminectomy groups, Student's t test or Mann-Whitney U test was used [26]. Statistical linear regression was performed to determine possible correlation relationships between the IRL of CGRP, p75NTR, Homer-1a, and 5-HT and the hypersensitivity threshold determined by the bilateral hot plate test. In this study, values were expressed as mean ± S.E.M. (the standard error of the mean), and statistical significance was set at p < 0.05.

General physical condition and signs suggestive of spontaneous pain after SCI
All animals lived through the experiment period except for one rat in the SCI group that was euthanatized due to a lower body skin lesion resulting from excessive licking possibly triggered by allodynia (note: another rat was removed from the study due to inadequate injury); light hematuria in several SCI rats resolved within 3-5 days. The group mean time to restore a reflex bladder in SCI rats was 4.3 ± 1.8 days (n = 12), which was typical for this model of injury [26] and much shorter than in rats with contusion SCI [27]. Control animals had neither postsurgical complication nor functional deficit. Thus, the group size of SCI and control animals was n = 12 and 10, respectively ( Fig. 1A, B).
Changes in body weight were recorded to monitor wellbeing of the rats. There was an anticipated degree of body weight loss within the first week following T10 compression. Specifically, relative to the pre-SCI level, the mean percentage of body weight reduction was 0% for the laminectomy group, and 4.2% for the SCI group (range: 0-8.1%; n = 12), which was far below 15% set as a criterion to remove animals from the study. All SCI rats recovered their body weight to levels before surgery after the first week p.i., and afterward, continuously gained weight at a rate normally observed in rats following similar surgical procedures (data not shown) [14,26].
To detect possible presence of spontaneous pain, a central component of clinical NP [5,7], signs suggestive of stress and/or pain, which included porphyrin staining, reduced grooming, licking or scratching of an intact body area, vocalization, etc., were scored based on housing checking chart records using a formula we developed Table 1 Antibodies for immunohistochemical (IHC) reactions 5-HT 5-hydroxytryptamine (serotonin), BDNF brain-derived neurotrophic factor, CGRP calcitonin gene-related peptide, DCX doublecortin, Fos the FBJ (Finkel-Biskis-Jenkins) murine osteosarcoma virus (v-Fos), GAD67 glutamic acid decarboxylase 67, GFAP glial fibrillary acidic protein, Iba1 ionized calcium binding adaptor molecule 1, iNOS inducible nitric oxide synthase, NeuN neuronal nuclear protein, p75NTR p75 neurotrophin receptor, SOX2 SRY (sex determining region Y)-box 2, TNFα tumor necrosis factor alpha (see Additional file 1: Table S1 for specifics including references). In the range of 0-38, a score ≥ 5 attained from a non-invoked animal suggested likely presence of pain. Whereas all control rats (n = 10) had 0 points during weeks 2-8 p.i., the SCI rats (n = 12) showed scores in a range of 5-10 and 2-5 in weeks 2-5 and 6-8, respectively, with the group median score consistently being 5, indicating that some mild degree of pain might exist since the average body weight gains were not disturbed.

Post-SCI reduction of hindlimb coordinated motor function Locomotion
Evaluation based on the BBB scale revealed marked deficits in hindlimb locomotion in rats after T10 SCI; in contrast, all rats after laminectomy showed no locomotor dysfunction (i.e., BBB score = 21; Fig. 1C). SCI rats demonstrated distinct reduction of hindlimb locomotor ability that was most profound at day 1 p.i. (Fig. 1C).
However, compared to the typical sign of spinal shock (i.e., complete flaccid paralysis: BBB score = 0) that occurs during the first 24 h after lower thoracic contusion and T10 severe compression (50 g × 5 min) [26,27], the SCI rats did not manifest spinal shock syndrome (i.e., group mean BBB score/day-1 p.i.: 3.1 ± 1.4; n = 12; Fig. 1C). Thereafter, hindlimbs showed a gradual recovery of locomotion that plateaued 3-4 weeks later. At the end of the study, the SCI group's mean BBB score was 16.1 ± 0.9 points (versus 21 ± 0 of the control group), showing a chronic deficit level that was less severe than that observed in the previous study using the same compression regimen (i.e., BBB scores of ~ 12) [14].

Incline plane test
Compared to the control group, the mean maximum degree where SCI rats could stabilize their body postures while facing-downward, mainly by the hindlimbs was significantly lower in 1 day p.i. (37.6 ± 2.0°/SCI versus a normal range of 55.7 ± 0.8°/control, p < 0.05; twoway repeated measures ANOVA with Sidak's post hoc test). As seen in the locomotor changes, incline plane performance of the SCI rats improved more over the first 3 weeks before plateauing around 4-8 weeks p.i. (Fig. 1D). At 8 weeks p.i., the SCI group's mean maximum incline plane degree was 49.3 ± 1.3°, which remained significantly lower than the 57.2 ± 0.9° of the control group (n = 12/SCI or 10/control; p < 0.05 to < 0.001; two-way repeated measures ANOVA with Sidak's post hoc test). In this study, the moderate T10 compression formula resulted in group average BBB scores and incline plane angles that were much closer to that of mild compression (20 g × 5 min) reported earlier [14,26]. Thus, the term of "moderate compression" was used to specify the physical regimen of the quasi-static compression per se (35 g × 5 min). This discrepancy was probably caused by subtle differences in surgical details such as laminectomy size produced by different operation performers.

Post-SCI abnormalities of sensory functions Neurological reflexes
The contact righting reflex was evaluated to assess the ability of the spinal cord to coordinate with the brainstem, peripheral afferent and efferent system, and neuromuscular junctions to correct abnormal body postures [14,26,[33][34][35]. Furthermore, postural (placing) and spinal reflexes were tested [14,26]. These data were presented as the percentage of rats in either group that had normal reflexes [14].
All SCI rats lost the ability to perform normal righting reflex ( Fig. 2A), proprioceptive positioning reflex of paw placing (Fig. 2B), and withdrawal response to hindlimb extension (Fig. 2C), at 1 day p.i. There were noticeable recoveries of the three categories of reflexes in the following weeks with 80% SCI rats showing normal righting and paw placing reflexes ( Fig. 2A, B), and extension reflex (Fig. 2C) by 3 and 4 weeks p.i., respectively. Conversely, withdrawal reflexes induced by brief pressuring (i.e., pressure withdrawal reflex; Fig. 2D) and pinching (i.e., nociception withdrawal reflex; Fig. 2E) of the hindpaw were detectable (despite severe reductions) 1 day p.i. (i.e., no spinal shock). Their recovery rates, however, were much slower and incomplete in comparison to those of the righting ( Fig. 2A), placing (Fig. 2B), or extension reflex (Fig. 2C). For example, by 8 weeks p.i., only ~ 33% rats with SCI showed a normal nociception withdrawal reflex (Fig. 2E), which was much lower than that of righting, placing, or extension reflex recovery (i.e., 100%; Fig. 2A-C), revealing a protracted abnormality of nociceptive signal-induced neurological reflexes in rats with T10 SCI.

Mechanical hypersensitivity
Mechanical allodynia, a representative symptom of NP, is a hypersensitivity triggered by innocuous stimuli similar to light touch. Clinically, the standardized VFT is used to detect a mechanical sensory threshold in the quantitative sensory testing (QST) for pain in patients [36]. In this study, mVFT, which emulated light touch, was performed to detect evoked mechanical hypersensitivity (Fig. 2F) [14,37]. Before SCI, the group withdrawal sensitivity threshold averaged 8.2 ± 0.6 g/control and 7.5 ± 0.4 g/ SCI in the forepaws (p > 0.05; Fig. 2G), and 8.6 ± 0.4 g/ control and 9.0 ± 0.8 g/SCI in the hindpaws (p > 0.05; Student's t test; Fig. 2H). In contrast to a stable array of sensitivity thresholds attained from the control group, at day 1 p.i., the mean withdrawal sensitivity threshold drastically increased to 11.1 ± 1.5 g and 45.8 ± 5.1 g in the forepaws and hindpaws of SCI rats, respectively (Fig. 2G, H), suggesting that there was severe loss of neurological functions close to that of spinal shock. Thereafter, the group mean withdrawal threshold, compared to the pre-SCI level and control values, significantly decreased in the forepaws (i.e., above-injury level allodynia), starting 2 weeks after SCI (4.7 ± 0.7 g/week 2 p.i., p < 0.05; 3.9 ± 0.5 g/week 5 p.i., p < 0.01; and 3.8 ± 0.9 g/week 8 p.i., p < 0.01; n = 12; Fig. 2G); the hindpaws of SCI animals also had significantly decreased mean withdrawal sensitivity threshold (i.e., below-injury level allodynia; 4.1 ± 0.6 g/ week 2 p.i., p < 0.01; 3.6 ± 0.4 g/week 5 p.i., p < 0.01; and 4.0 ± 0.3 g/week 8 p.i., p < 0.01; n = 12; Fig. 2H). Lastly, atinjury level mechanical allodynia measured in the dorsal dermatomes adjacent to the T10 injury epicenter (Fig. 2I) exhibited a comparable pattern of significant augmentation of hypersensitivity, compared to the control group (0.8 ± 0.3 g/week 2 p.i., p < 0.01; 0.5 ± 0.2 g week 5 p.i., p < 0.01; and 0.4 ± 0.1 g week 8 p.i., p < 0.01; n = 12/SCI or 10/control; two-way repeated measures ANOVA with Sidak's post hoc test; Fig. 2I). For all three levels tested, there were no significant changes in withdrawal sensitivity thresholds in the laminectomy control group. These data demonstrated that SCI rats displayed subacute and chronic hypersensitivities to mechanical stimuli above-, below-, and at-injury level.

Thermal hypersensitivity
Thermal hypersensitivity threshold is an important parameter in the QST for diagnosing thermal allodynia, another important symptom of clinical SCI NP [38][39][40][41]. SCI and control rats first underwent the bilateral hot plate test for which the pre-SCI and pre-laminectomy mean hindlimb latency was 8.8 ± 0.8 s (n = 12) and 8.0 ± 0.9 s (n = 10; p > 0.5, Student's t test), respectively (i.e., baseline values; Fig. 2J). While the control group showed a steady mean response latency, the group mean latency increased dramatically to 19.1 ± 3.1 s in the injured rats at day 1 p.i., relative to the baseline value (i.e., 8.8 ± 0.8 s), indicating a transient severe reduction of neurological function during the first 24 h p.i. Subsequently, the group mean latency of SCI animals gradually decreased, with the average threshold becoming significantly lower (i.e., ↑thermal hypersensitivity) than the baseline values and those of the control group, starting in week 3 p.i. (Fig. 2J; n = 12/SCI or 10/laminectomy; p < 0.05; two-way repeated measures ANOVA with Sidak's post hoc test).
In the more sensitive unilateral hindlimb hot plate test [39], although an overall resemblance to the bilateral hot plate test data was observed, this test detected significantly reduced group mean latencies 1 week earlier, starting day 14 p.i., which lasted till the end of the study, compared to the control group ( Fig. 2K; n = 12/SCI or 10/control; p < 0.05; two-way repeated measures ANOVA with Sidak's post hoc test). Furthermore, the SCI group mean threshold of this test trended even lower than the bilateral hot plate test results (Fig. 2K), confirming the test was more sensitive.

Histopathological impacts of moderate T10 compression
Whereas no discernible lesions were found in the laminectomy control tissues, all injured spinal cords exhibited apparent damages in the white matter (dorsal, lateral, and ventral funiculi) and gray matter (dorsal and ventral horns, and intermediate and central regions) at the injury epicenter, forming cavities and lesion areas that further extended rostrally and caudally to demarcate the lesion volume (Fig. 3A). Epicenter coronal sections also contained a high number of basophilic inflammatory cells (i.e., subcellular structures containing nucleic acids stained dark blue/violet by hematoxylin) and intracavity loose webs of non-neural tissues (Fig. 3A). Farther away from the epicenter, sections sampled from T8-9 spinal cord that was ~ 5 mm rostral to the compression site (Fig. 3B), exhibited discernible demyelination (i.e., hypostained by solvent blue) and infiltration of inflammatory cells in both the white matter and gray matter (Fig. 3C).
Measurements of digital images of representative coronal sections at the injury site and 1-3 mm rostral and caudal to it, demonstrated that relative to the control group, T10 compression significantly reduced the mean residual white matter (Fig. 3D) and gray matter areas (Fig. 3E) at the epicenter, and in loci 1 and 2 mm adjacent to it (p < 0.05; two-way repeated measures ANOVA with Sidak's post hoc test; n = 7/group). Taken together, the morphological evidence showed chronic neuroparenchyma loss and lesions, and presence of inflammatory cells in the epicenter and regions bidirectionally adjacent to it after T10 compression [14,26].

Chronic NIF, NTM, NML, and NPL changes at multiple nuclei/regions of the CNS in rats with SCI NP
Our previous work demonstrated that lumbar and cervical NIF co-existed with NP-like behaviors in the early chronic phase of SCI [14,26]. However, whether NIF after a T10 injury may extend into other thoracic cord levels such as T8 where more sympathetic preganglionic neurons reside [27], and different brain centers involved in pain signaling relay and regulation remained to be systematically investigated [14,17,42,43].

Changes of NIF, NTM, NML, and NPL markers in injured spinal cords
To assess NIF responses in T8 neuroparenchyma, IRL of GFAP (astrocyte activation and reactive astrogliosis), Iba-1 (microglia/macrophage activation), TNFα (proinflammatory cytokine), and/or iNOS (proinflammatory polarization marker of microglia/macrophages and an inflammation mediator) expressions were evaluated in spinal cord sections sampled from 5 mm rostral to the injury epicenter (SCI group) or T10 level (i.e., laminectomy site; control group) ( Fig. 4A; see Fig. 4B for the labeling of the framed areas that were examined: corresponding higher magnification images were in Fig. 4C . Injured spinal cord sections at and around the epicenter exhibited damages in the white matter and gray matter (e.g., cavities and lesion areas). The injured tissues also contained a large number of basophilic inflammatory cells (stained by hematoxylin) and intra-cavity loose webs of non-neural tissues. B, C T8 spinal cord sections that were ~ 5 mm rostral to T10 injury site (B; framed areas were magnified in C), had apparent demyelination (i.e., hypostained by solvent blue) and infiltration of inflammatory cells in both white matter and gray matter (C). D, E Area measurement of representative coronal sections at the injury site (i.e., 0 mm) and 1-3 mm rostral and caudal to it, revealed that compared to the control group, T10 compression significantly reduced the mean residual white matter (D) and gray matter areas (E) at the epicenter, and in spinal cord loci 1 and 2 mm adjacent to it (p < 0.05; two-way repeated measures ANOVA with Sidak's post hoc test; n = 7/ group) Next, expression levels of calcitonin gene-related peptide (CGRP), a main neurotransmitter released from the C and Aδ sensory fiber terminals onto the substantia gelatinosa neurons, which facilitates NP/pain development [44,45], and p75 neurotrophin receptor (p75NTR), a key receptor to regulate peripheral and central nociceptive neurons [46], were evaluated with IHC assays of T8 spinal cord sections (SCI group) or at T10 level (control). Relative to control tissues ( . For SCI NP-triggered NPL and NML alterations [14], IHC analysis uncovered significantly increased group mean IRL of Homer-1a, a molecular marker for DH neuronal plasticity (Fig. 5E, G) [14] and serotonin (5HT, a neuromodulator; Fig. 5E 1 , G 1 ) in RL-I and II, relative to the control group ( Fig. 5F; p < 0.05, n = 4/group, Student's t test).
Lastly, to determine possible functional impacts associated with expression changes of the afore-described molecules p.i., statistical linear regressions were conducted. The results showed that IRL of CGRP and p75NTR were negatively correlated with the thermosensitivity threshold (unit: second) determined by the bilateral hot plate test (CGRP: p = 0.02, R 2 = 0.83, Fig. 5K; p75NTR: p < 0.01, R 2 = 0.94, Fig. 5L) for the SCI group. Moreover, changes in IRL of Homer-1α and 5HT also correlated negatively with the thermosensitivity threshold derived from the bilateral hot plate test in animals with SCI (Homer-1α: p < 0.01, R 2 = 0.72, Fig. 5M; 5HT: p < 0.01, R 2 = 0.85, Fig. 5N). These data suggested that increased DH expressions of CGRP and p75NTR might have worsened SCI NP, which could have triggered stronger NPL and NML responses (e.g., higher expressions of Homer-1α and 5HT, respectively, in RL-I and II of DH).

Chronic NIF, neuronal hyperactivity, and serotonin reduction in the brainstem
Situated in the medulla oblongata at the junction of the cervical spinal cord and brainstem, neurons in the GrN (Fig. 6A) relay somatosensory information from the lower half of the body and the legs to the thalamus and participate in maintaining NP [47,48]. In this study, significantly higher group mean IRL of Iba-1 (Fig. 6B, D) and TNFα (Fig. 6B 1 , D 1 ) presented in brainstem coronal sections containing GrN of SCI rats compared to those of the control group (Fig. 6C). IHC double stains of the same level sections showed significantly elevated mean IRL of GFAP (Fig. 6E, G 1 ) and iNOS (Fig. 6E 1 , G) in the SCI rats relative to the control group ( Fig. 6F; n = 4/ group; p < 0.05, Student's t test). Because c-Fos expression in secondary sensory neurons in the GrN could be augmented by electrical or nerve injury stimulation of c-fibers to emulate nociceptive afferent signals [49], numbers of cFos+ neurons in GrN were quantified via IHC colocalization of cFos, and nuclear markers of NeuN and DAPI. The mean number of neurons producing cFos was significantly higher in SCI animals than that of controls (  Fig. 6K, n = 4/group; p < 0.05, Student's t test). Orthogonal slicing confirmed co-localization of cFos, NeuN, a highly specific marker of neuronal nuclei (Fig. 6J), and DAPI, a standard nuclear counterstain (Fig. 6J 1 ).
Positioned in the rostral ventromedial medulla (RVM) of the brainstem is the nucleus raphe magnus (NRM or RMg; Fig. 6L), a serotonergic nucleus (Fig. 6L 1  receives inputs from the PAG (see below) and mainly projects to the spinal cord DH [50]. In the SCI tissue sections, significantly increased group average IRL of GFAP was observed in NRM, compared to the control group ( Fig. 6N 1 /SCI versus Fig. 6N 2 /control; note: GFAP was mainly produced by hypertrophic astrocytes in injured spinal cords; comparing inset image in Fig. 6N 1 to that of Fig. 6N 2 ). In contrast, SCI NRM had markedly reduced  suggested that T10 compression induced regional/systemic NIF and heightened DH demand of 5HT (Fig. 5E 1 , G 1 ), which might conjointly diminish NRM production and storage of 5HT. Farther rostral to GrN and NRM, located bilaterally in the dorsolateral pons, are the lateral PBN (LPBN: Fig. 7A) that disseminate sensory information (e.g., temperature, pain, etc.) to forebrain structures (e.g., the thalamus, hypothalamus, and extended amygdala) with the involvement of their CGRP+ neurons in the development of NP [50][51][52]. SCI LPBN displayed significantly elevated group mean IRL of co-stained Iba-1 (Fig. 7B, D)/TNFα (Fig. 7B 1 , D 1 ) and iNOS (Fig. 7E 1 , G)/GFAP (Fig. 7E, G), compared to control LPBN (Iba-1/TNFα: Fig. 7C; iNOS/ GFAP: Fig. 7F; n = 4/group, p < 0.05, Student's t test). Furthermore, IHC images showed typical morphological features of activation and aggregation of microglia and astrocytes in the LPBN area after SCI, corroborating with increased IHC detection of Iba-1, TNFα and iNOS, and GFAP in these cells (Fig. 7B, B 1 , E 1 , and E, relative to Fig. 7C, F), respectively. SCI boosted the activity of NeuN+ (Fig. 7H) . This data suggested that pathophysiological activation of the LPBN CGRP neurons coexisted with NIF and NP in rats after T10 compression.

Chronic NIF and higher GAD67 level in GABAergic neurons in the PAG
The PAG (also termed the central gray), especially the ventral lateral PAG (VLPAG: Fig. 7M) in the midbrain, is a major descending modulation center of pain [53].

Chronic NIF and altered NSC activity in the subcortical structures
The subcortical structures primarily consist of the limbic system (i.e., the hypothalamus, the amygdala, the thalamus, and the hippocampus), basal ganglia, and olfactory bulb [54,55], which play roles in chronic pain-induced  's t test). IHC images showed morphological features of aggregation of activated microglia and astrocytes in LPBN after SCI, corroborating with increased detection of Iba-1, TNFα and iNOS, and GFAP in these cells (B, B 1 , E 1 , and E, relative to controls in C and F). T10 compression heightened the activity of NeuN+ (H)/CGRP+ (I) neurons in the LPBN compared to the control group (J; confocal analysis in K/SCI and K 1 /control), which was demonstrated by significantly higher numbers of cFos+ cells co-expressing CGRP in SCI LPBN (statistics in L; n = 4/group, *p < 0.05, Student's t test). The ventral lateral periaqueductal gray (VLPAG: Bregma −7.80 mm; M) in the midbrain of the SCI group showed higher mean IRL of GFAP and Iba1/TNFα co-staining (N and P 1 /P 2 /P 3 , respectively) than the control group (N 1 /GFAP; P 4 /Iba1 and TNFα); the difference was statistically significant (O/GFAP, n = 4/group, ***p < 0.001; Student's t test; Q/Iba1 and TNFα, *p < 0.05; Mann-Whitney U test; n = 4/group). Under higher magnification, SCI VLPAG manifested extensive reactive gliosis (e.g., morphology of hypertrophic astrocytes: N inset), relative to the control tissue (N 1 inset). Activated microglia (i.e., swollen ramified morphology and expressions of Iba1/P 1 inset and TNFα/P 2 inset (merged images: P 3 inset) densely populated VLPAG of SCI animals only (relative to laminectomy controls: P 4 and inset). Importantly, discernably elevated GAD67 expression scale existed in the VLPAG of the SCI group (R 1 ) than the control group (R 2 ). Confocal z-stack imaging with orthogonal optical slicing depicted a typical profile of GABAergic neurons with GAD67 immunostaining in the neuronal somas and neurites (R 3 ). The mean IRL of GAD67 in the SCI VLPAG was significantly higher than that of the control group (S; n = 5/group, **p < 0. depression, anxiety, perception, cognition, and memory [55][56][57][58]. Our investigation of the hippocampal formation (Fig. 8A) revealed that the dentate gyrus (DG) cortex of the SCI animals had a qualitatively higher level of astrocytic GFAP (Fig. 8B 1 ) compared to laminectomy controls (Fig. 8B 2 ). Due to the definitive difference in GFAP IRL observed (i.e., cytoarchitecture of Fig. 8B 1 inset versus that of Fig. 8B 2 inset), no statistical comparison was made. Also, there were more areas occupied by Iba-1 expressing activated microglia, mostly in the neurogenic subgranular zone (SGZ; Fig. 8C 1 and inset), in the DG of the SCI animals than the controls (Fig. 8C 2 ); the difference was statistically significant (Fig. 8C 3 ; p < 0.01; Student's t test; n = 4/group). Importantly, the SCI DG sections were more compactly settled by nuclear Sox2+ (Fig. 8D 1 ) and cytoplasm nestin+ NSCs (Fig. 8D 2 ; DAPI nuclear counterstain in Fig. 8D 3 ; merged image in Fig. 8D 4 ), with some early phase NSCs expressing nuclear Sox2 only (see confocal orthogonal slices in Fig. 8D 5 ). Conversely, fewer NSCs presented in the control section (Fig. 8E 1-4 ), rendering the mean IRL of Sox2 and nestin significantly lower than the SCI group ( Fig. 8F; p < 0.05; Mann-Whitney U test; n = 4/group). Further, tissues of SCI animals (Fig. 8G 1-3 ), relative to controls (Fig. 8G 4 ), had significantly elevated expression of BDNF (Fig. 8G 1 ) in cells that were mostly negative for doublecortin (DCX), a marker for migrating NSCs (Fig. 8G 2-5 ). Most BDNF+ cells showed features of activated astrocyte or microglia (comparing Fig. 8G 2 to Fig. 8B 1 and C 2 ); however, some DCX+ NSCs also produced BDNF (i.e., co-stained cells in Fig. 8G 3 : optical slices in Fig. 8G 5 ). The mean IRL of BDNF, not DCX, was significantly different between the two groups (Fig. 8H, p < 0.05, Mann-Whitney U test; n = 4/group).
The BLA (Fig. 8I) is an important member of the fear extinction circuitry [59]. Neurotensin receptor 2 (NTR2) expressing neurons in the BLA were recently found to play a crucial role in fear inhibition [60]. The BLA tissue of the SCI group had an average IRL of NTR2 or GFAP that was significantly lower or higher, respectively, than the control group (p < 0.001 and 0.01, respectively; Student's t test; n = 4/group; Fig. 8J). The BLA of SCI animals (Fig. 8K), compared to controls (Fig. 8L), expressed discernibly less NTR2 (Fig. 8K 1  Noticeably, IHC morphological profiles showed that NTR2 was mostly expressed in neuronal bodies and neurites (i.e., GFAP− cells); however, some GFAP+ astrocytes also expressed NTR2 (i.e., yellow cells in Fig. 8K 3 , L 3 insets) as reported previously [61].

Discussion
Chronic pain is one of the most frequent and debilitating complications of SCI [62][63][64][65]. NP, defined by the International Association for the Study of Pain (updated Dec. 14, 2017) as "pain caused by a lesion or disease of the somatosensory nervous system", affects more than half of people who have experienced SCI [2,3]. Conventional therapeutics such as lithium, gabapentin, and tramadol, which are designed to tackle a single target of neurotransmission, often exhibit short term efficacy with considerable side effects [66][67][68][69]. To date, reliable management of clinical SCI pain, especially, NP, remains an (See figure on next page.) Fig. 8 Neuroinflammatory responses, changes of NSC activity in HPC, and reduction of NTR2 expression in BLA after SCI. The DG (in red rectangle) of the hippocampal formation (Bregma − 4.80 mm; A) of a SCI rat showed markedly augmented reactive astrogliosis assessed by GFAP IHC stain (B 1 and inset), relative to a control image (B 2 and inset). Moreover, the mean IRL of Iba1 (red) expressed by hypertrophic microglia (C 1 inset: lower left column) concentrated mostly in the neurogenic SGZ of the DG in SCI rats (C 1 inset of upper left column: Iba1 images in SGZ versus those in Gr) was higher than the controls (C 2 ); the difference was statistically significant (C 3 ; **P < 0.01; Student's t test; n = 4/group). SCI DG sections exhibited much more nuclear SOX2 (green, D 1 ) and cytosol nestin immunostains (red, D 2 ; DAPI in D 3 ) compared to those of control tissue (E 1-3 ). The merged z-stack images (D 4 ) showed that some NSCs had SOX2 (green) and DAPI (blue) co-stained nuclei (cyan in D 5 ). The differences between the IRL means of the two groups were significant (F; *P < 0.05; Mann-Whitney U test; n = 4/group for both SOX2 and nestin). The SCI HPC sections (G 1-3 ), relative to controls (G 4 ), exhibited significantly stronger BDNF immunosignal (green, G 1 ) in cells that were mostly negative for DCX (red, G 2 ). Some DCX+ NSCs also expressed BDNF (yellow cells in G 3 ). The mean IRLs of BDNF, not DCX, were significantly different between the two groups (*P < 0.05 or ns, Mann-Whitney U test, respectively; n = 4/group; H). SCI-triggered neuroinflammatory impacts were evaluated in the BLA (Bregma − 2.30 mm; I). Mean IRL of NTR2 (red) or GFAP (green) in the SCI group (K 1-3 ) was significantly lower or higher than the control group (L 1 -L 3 ), respectively (J; ***P < 0.001 for NTR2; **P < 0.01 for GFAP; Student's t test; n = 4/group). The NTR2 immunostain was mainly located in the cytosol fractions of GFAP− cells, suggesting the cells were BLA interneurons (see morphologic differences between NTR2+ cells/L 4 and GFAP+ astrocytes/K 4 ). Scale bars:100 µm except for K 4 and L 4 where they were 20 µm. BLA basolateral amygdala, DCX doublecortin, DG dentate gyrus, ec external capsule, Gr granular layer of DG, HPC hippocampus, Mo molecular layer of DG, ns not significant, NSC neural stem cell, NTR2 neurotensin receptor type 2, Po polymorphic layer of DG (i.e., hilus), SGZ subgranular zone of DG unmet medical demand [62,66]. The situation is partly attributable to a lack of a comprehensive understanding of the systemic scale, dimension, and diversity of pathophysiological mechanisms of NP [2,62,66]. Clinical NP manifests as spontaneous pain and/or evoked pain (with less frequency); it occurs as modalityspecific sensory gain or loss (e.g., allodynia, hyperpathia and hyperalgesia, or anesthesia dolorosa). Thus, NP is difficult to define in experimental settings, and therapeutics that are effective in laboratory studies often do not translate directly to patients [23]. Animal models of evoked limb withdrawal are commonly used to detect allodynia and hyperalgesia-like hypersensitivities, but they may reflect only the evoked pain component in patients [70]. Moreover, there still lacks a standard model to measure "spontaneous pain" despite using the conditioned place preference (CPP) assay, as CPP typically detects the rewarding and aversive (i.e., reinforcing) effects of drugs. The assessment of spontaneous pain in rodents has become a multiplex subject of extensive debate [71].
In theory, there are multiple potential biomarkers in pain models that may enable improving clinical relevance predication [72]. In this study, the application of mVF, hot plate, and spinal reflex tests is based on the principle of aligning sensory measurements for animal models with current methods of clinical sensory assessment [73]. On this basis, we propose that the evoked mechanical and thermal responses plus the sensorimotor, histopathological, NIF, NML and NPL parameters for lab models of pain or SCI can be redeployed as sensory profiling tools to generate a multidimensional biomarker profile of NP (MBPN) after neurotrauma; the integration of spontaneous pain appraisal via quantifying pain-related physical/ behavioral signs (e.g., porphyrin staining, vocalization, etc.) may further improve clinical relevance of this formula [74]. Our MBPN-based novel approach, which does not merely measure "pain" after experimental SCI, may improve translational strength of the data for future research of NP and analgesic development [24,75].
NP occurs from a few days to several months after clinical SCI, during which one of the secondary injury processes is NIF, hallmarked by reactive gliosis, microglia/ macrophage activation, and heightened production of proinflammatory cytokines from residential and invading inflammatory cells [76][77][78]. These events, acting alone or in consortium, have been identified as promoters of NP [15,[79][80][81]. Here, we investigated whether a long-term coexistence of NP and multilocus NIF might present in a rat model of T10 compression (Fig. 1). This SCI model was previously established in our laboratory to emulate clinical quasi-static insults to the spinal cord (e.g., injury resulting from vertebral fracture, abscess, herniated intervertebral disk, arthropathy, or tumor) [26,82]. The T10 SCI model produced above-, at-, and belowinjury level mechanical hypersensitivity in all SCI rats (Fig. 2), in contrast to clinical SCIs that affect about 50% of individuals and show above-level allodynia in the least prevalence [83,84].
Whereas our finding is consistent with laboratory data published by others [85][86][87], the field does not appear to have unified mechanistic explanations for these discrepancies. Conceivably, compared to humans, rodents could have much stronger sensitivity and response (including those of NIF/NTM/NML/NPL) to nociceptive experiences, which as a pivotal survival mechanism may underlie their higher rate of NP after SCI. Further, discernibly more wide dynamic range (WDR) neurons were found in DHs 2-3 segments above injury site in the allodynic rats [85], suggesting that rodents, relative to humans, may have a higher amplitude of maladaptive neuroplasticity in DH sensory neurons. The higher prevalence of abovelevel mechanical allodynia in SCI rats could be attributable to this increased proportion of WDR neurons. Importantly, it has been reported that p.i. above-level allodynia in humans may not be due to the SCI itself, but rather linked with concurrent peripheral nerve injury (PNI) [83,84], and in SCI models, this could be triggered by peripheral nerve sensitization in response to the secondary injury process [88,89].
As observed before in the same and other SCI models, NIF at the non-treated epicenter and the lumbar and cervical spinal cord stayed active during the subacute and early chronic phases p.i., with most microglia exhibiting a pro-inflammatory phenotype to worsen p.i. destructive sequelae [14,26,[76][77][78]. Accordingly, compared to the laminectomy operation, T10 compression, besides causing sensorimotor and histopathological damages (Figs. 2 and 3), markedly elevated inflammatory responses in (1) RL-I and II of the DH where the peripheral nociceptive signals from dorsal root ganglion (DRG) neurons were transmitted to the secondary sensory neurons (note: for our focus on the CNS, DRGs were not analyzed); (2) the lateral and ventral funiculi that housed the spinothalamic pathway; (3) the DC containing the touch and kinesthesia primary pathways that synapsed in the GrN and cuneate nucleus, and (4) the spinoparabrachial pathway in the lateral and dorsal columns that relays in the PBN, involved in the affective component of pain (Fig. 4) [90]. In these sensory nuclei, chronic NIF characterized by microglial and astrocytic activation plus augmented expression of pro-inflammatory cytokines and iNOS (iNOS+ glia are proinflammatory) coexisted with behavioral abnormalities of sensorimotor reflex, evoked mechanical and thermal hypersensitivity (Figs. 4, 5, 6, 7 and 8), and signs suggestive of spontaneous pain in the SCI group only. Also assessed was the IHC level of CGRP, a key neurotransmitter released from the C and Aδ afferent fiber terminals for pain and temperature sensation onto the DH substantia gelatinosa neurons [44,45] and p75NTR, a main modulator of DRG and DH RL-I and II neurons for nociceptive transmission [46]. The significantly increased expression of the two molecules in the DRG neuronal afferent terminals and DH neurons of the SCI rats, relative to the controls, indicated that these neurons were hypersensitive, which might have partly ignited and upheld the NP-like behaviors and signs. This conclusion was supported by the statistical linear regression data that demonstrated significant reverse correlations between IRL of CGRP and p75NTR, and thresholds of the bilateral hot plate test (Fig. 5).
We earlier reported that sensorimotor abnormalities in the same T10 SCI model were associated with perturbated expressions of Homer-1a (an immediate-early gene that is increased by synaptic activity) and 5HT in the distal spinal cord [14,91]. Changes of these two factors have been linked to pain-induced NPL and sensorimotor NML, respectively [14,92]. The group average IRLs of Homer-1a and 5HT were significantly higher in the SCI DH than the control DH. Moreover, the IRLs of Homer 1a and 5HT were in significant negative correlation with the bilateral hot plate test thresholds of SCI rats (Fig. 5). The results collectively suggested that the DH neural network under this scale of hypersensitivity was still in a state attempting to modulate the elevated nociceptive input via enhancement of Homer-1a and 5HT expression. Since treating rats 3 weeks after the same type of SCI with huperzine A, a multimodal neuromodulation drug, markedly impeded NIF, DH expression of Homer-1a, and NP-like behaviors [14], future therapeutic development should target these neurodynamic events to manage SCI NP [50,[91][92][93].
Lumbar NIF responses, such as activation of astrocytes and microglia in the lumbar DH after T13 hemisection [93] or L5 microglia activation/↑TNFα and IL1β post-midthoracic SCI [94], have been associated with below-injury level allodynia; in addition, supraspinal NIF and NPL reactions were found in animals with SCI NP [94][95][96]. We thereby decided to systematically examine possible presence of NIF in major sensory relay and modulation centers concerning allodynia, nocifensive, and affective processes in the brainstem and brain subcortical structures. In the brainstem, GrN neuronal sensitization has been correlated with development of NP and tactile allodynia in the lower limbs [97,98]. Here, IRL of the immediate-early gene cFos, a marker of painrelated DH neuronal activation [14,99], was significantly higher in the GrN neurons (Fig. 6) in the context of activated microglia and astrocytes residing in the same region. The changes were coupled with NP-like behaviors of the SCI rats 8 weeks after SCI. Located farther rostral in the brainstem are LPBN neurons that are involved in the affective component of pain by transmitting visceral and somatic sensory information to the forebrain [51]. Published data suggested that LPBN CGRP+ neurons could transmit nociceptive signals via innervation of the paraventricular thalamic nucleus and central amygdaloid nucleus [51,52]. Rats with chronic hyperalgesia post-T10 compression showed elevated group average scales of astrocytic and microglial activation, IRL of proinflammatory cytokines, and numbers of cFos+/CGRP+ neurons in the LPBN (Fig. 7), reconfirming a possible interaction between PBN NIF, LPBN CGRP+ neuronal hyperactivity, and NP in the chronic phase of SCI.
The descending pain modulatory system (DPMS) circuits consist of the anterior cingulate cortex, amygdala, anterior insula, middle frontal gyrus, RVM, and PAG [100]. The NRM, residing inside the RVM of the brainstem with input from PAG, is the principal source of CNS serotonergic projections including those to the DH, to biphasically modulate nociceptive control during adulthood [101]. However, there has essentially been no published data about possible connections between NRM NIF, altered 5HT expression in NRM or DH, and NP after SCI. In our investigation, significantly higher group mean GFAP IRL was found in hypertrophic astrocytes within NRM of SCI rats only (Fig. 6). Conversely, post-SCI NRM 5HT IRL was markedly reduced (Fig. 6), which was likely caused by chronic NIF [102], as well as surged supply demand of 5HT from RL-I and II of DH (Fig. 5) due to ongoing SCI NP [101]. Correspondingly, the VLPAG of SCI animals had significantly higher IRL of NIF markers (i.e., ↑GFAP, ↑Iba-1, and ↑TNFα) and expression of GAD-67 (Fig. 7), which metabolizes glutamate into GABA (an inhibitory neurotransmitter) and is a marker of GABAergic neurons. As a consequence, the reduced capacity of NRM serotonin and raised PAG GABA in chronic SCI could attenuate descending 5HTbased pain modulation and endogenous opioid-mediated analgesic effect, respectively, as well as conjointly disrupt the PAG-NRM-DH pain suppression pathway in DPMS to promote NP [100,103,104].
A feedforward inhibition enabled by BLA via a prefrontal cortex-PAG-NRM-spinal cord pathway for descending noradrenergic and serotoninergic modulation of afferent pain signals, plays a pivotal role in preventing mechanical and thermal hypersensitivity [105]. Furthermore, the spinoparabrachial pathway projects to BLA via PBN for sensory functions [106], and most BLA neurons innervate the hippocampus and neocortex to form bidirectional communications involved in emotion-related memory concerning depression [107,108]. Indeed, a population of fear-off neurons expressing NTR2 has been identified in BLA for anxiety regulation [60,109], and neurotensin and NTR2 were shown to directly modulate pain resulting from inflammatory stimuli [110]. Our examination of BLA revealed that SCI, relative to laminectomy alone, significantly heightened reactive gliosis (↑GFAP) but lowered neuronal IRL of NTR2 in the BLA (Fig. 8). The data suggested that T10 compression might interfere with BLA function by inducing NIF and weakening the function of fear-off neurons, which could contribute to development of NP and pain-triggered mood disorders [111]. Fig. 9 Summary diagram. The data of this study (see details in Figs. 1, 2, 3, 4, 5, 6, 7 and 8) suggested that T10 compression in young adult female rats resulted in coexistence of chronic neuropathic pain (NP) and multilevel neuroinflammation, neurotransmission, neuroplasticity, and neuromodulation responses along the neuroaxis. Such injury-induced neural pathophysiological events in main spinal cord and brain sensory/ pain processing centers have generated novel biomarkers as potential mechanistic underpinnings of SCI NP. The information can be used to multidimensionally profile experimental NP to enhance clinical relevance of pain modeling for therapeutic development For post-PNI chronic pain-evoked cognitive deficit, memory dysfunction, anxiety, and depression, NIF and neurogenic changes in the hippocampus, the key structure to process memory, learning, and emotion, have been identified as primary operators [112][113][114][115]. Yet, no published data has been available concerning associations between lower thoracic SCI, NP, and alterations of hippocampal NIF and neurogenesis. In this study, T10 compression, relative to laminectomy, significantly increased NIF and NSC presence in the hippocampus of rats with chronic SCI NP (Fig. 8). However, the two groups had comparable IRLs of hippocampal DCX, a marker of migratory NSCs, indicating that SCI caused neurogenic abnormalities beyond NSC proliferation (e.g., ↓migration of newly generated NSCs). This together with the heightened expression of BDNF, which appeared to be produced by the activated glia and NSCs (Fig. 8), could trigger hippocampal synaptic disorders as seen in a PNI model of NP to underlie pain-induced cognitive, mood, and memory problems [114,116]. In fact, down-regulation of adult hippocampal NSC proliferation reversibly blocked persistent pain, whereas increased NSC activity lengthened pain behaviors in murine models of PNI NP and hindpaw inflammatory pain [115].

Conclusions
To our best knowledge, this is the first study where NP-like behaviors and multilevel NIF, NML, and NPL changes in the CNS have been comprehensively characterized following a focal lower thoracic spinal cord trauma (Fig. 9). Limitations of this work included a single timepoint evaluation of NIF at 8 weeks p.i., no pharmacological or genetic manipulation to determine any causal relationship between hyperalgesia and specific biomarkers, no use of video-based analysis of persistent pain (e.g., the Grimace Scale) [117], and no inclusion of male animals despite the fact that female rats have conventionally been used in SCI modeling for better post-care results and more efficient development of hypersensitivity [118]. These aspects and the relationship between p.i. NP and possible NIF in the somatosensory cortex (which is beyond the scope of the current study) should be investigated in the future. Overall, the data have provided systematic biomarkers as mechanistic leads to profile SCI NP. We concluded that this model of SCI and its outcome measures have established a pertinent regimen to multidimensionally investigate p.i. NP, which may facilitate identification and translation of novel therapeutic targets for devising multimodal treatments of NP after SCI.


s2fieldsofstudy: Biology Chemistry

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High-Throughput Chemical Screen Identifies a 2,5-Disubstituted Pyridine as an Inhibitor of Candida albicans Erg11

ABSTRACT Fungal infections contribute to over 1.5 million deaths annually, with Candida albicans representing one of the most concerning human fungal pathogens. While normally commensal in nature, compromise of host immunity can result in C. albicans disseminating into the human bloodstream, causing infections with mortality rates of up to 40%. A contributing factor to this high mortality rate is the limited arsenal of antifungals approved to treat systemic infections. The most widely used antifungal class, the azoles, inhibits ergosterol biosynthesis by targeting Erg11. The rise of drug resistance among C. albicans clinical isolates, particularly against the azoles, has escalated the need to explore novel antifungal strategies. To address this challenge, we screened a 9,600-compound subset of the University of Tokyo Core Chemical Library to identify molecules with novel antifungal activity against C. albicans. The most potent hit molecule was CpdLC-6888, a 2,5-disubstituted pyridine compound, which inhibited growth of C. albicans and closely-related species. Chemical-genetic, biochemical, and modeling analyses suggest that CpdLC-6888 inhibits Erg11 in a manner similar to the azoles despite lacking the canonical five-membered nitrogen-containing azole ring. This work characterizes the antifungal activity of a 2,5-disubstituted pyridine against C. albicans, supporting the mining of existing chemical collections to identify compounds with novel antifungal activity. IMPORTANCE Pathogenic fungi represent a serious but underacknowledged threat to human health. The treatment and management of these infections relies heavily on the use of azole antifungals, a class of molecules that contain a five-membered nitrogen-containing ring and inhibit the biosynthesis of the key membrane sterol ergosterol. By employing a high-throughput chemical screen, we identified a 2,5-disubstituted pyridine, termed CpdLC-6888, as possessing antifungal activity against the prominent human fungal pathogen Candida albicans. Upon further investigation, we determined this molecule exhibits azole-like activity despite being structurally divergent. Specifically, transcriptional repression of the azole target gene ERG11 resulted in hypersensitivity to CpdLC-6888, and treatment of C. albicans with this molecule blocked the production of the key membrane sterol ergosterol. Therefore, this work describes a chemical scaffold with novel antifungal activity against a prevalent and threatening fungal pathogen affecting human health, expanding the repertoire of compounds that can inhibit this useful antifungal drug target.

F ungal pathogens are a significant threat to human health, infecting over a billion people globally and resulting in 1.5 million deaths annually (1). Candida albicans is a natural constituent of the human microbiota; however, in immunocompromised individuals it can become pathogenic and cause serious systemic infections with resulting mortality rates of up to 40% (1)(2)(3). Currently there are only three major classes of antifungals available to treat these systemic infections: azoles, echinocandins, and polyenes. The azoles are a class of five-membered, nitrogen-containing heterocyclic compounds that inhibit the biosynthesis of the essential cell membrane component, ergosterol, by targeting the lanosterol demethylase enzyme encoded by ERG11. This results in the production of aberrant sterols, which exert membrane stress and ultimately halt yeast growth (4). Azoles are the most widely deployed antifungal drug class, as they are broad spectrum, have a good safety profile, and are orally bioavailable (4). However, their extensive deployment, paired with fungistatic mode of action, has spurred the emergence of drug-resistant isolates, which threatens their utility (4). Thus, it is crucial to explore and develop new antifungal treatment strategies.
Chemical screen identifies CpdLC-6888 as a compound with activity against C. albicans. To identify compounds with novel antifungal activity against C. albicans, a high-throughput screen of a 9,600-chemical subset of the University of Tokyo's "Core Library" was performed. This subset includes molecules that exhibit more drug-like features based on structural and physical properties while also enriching for structural diversity. Four distinct hit compounds were identified that inhibited growth of C. albicans by more than 80% relative to drug-free controls ( Fig. 1A and B), three of which (CpdLC-2800, CpdLC-376, and CpdLC-6888) were procurable from an independent supplier. The potency of each compound against C. albicans was assessed via a standard doseresponse assay in which growth of cells was measured in a 2-fold dilution series of each compound (Text S1) (5). The minimum inhibitory concentration (MIC) that reduced growth by 90% (MIC 90 ) was calculated, with CpdLC-6888 emerging as the most potent compound exhibiting a MIC 90 value of 3.13 mM in yeast extract-peptone-dextrose (YPD) (Fig. 1C) and a MIC 70 value of 1.56 mM in RPMI (data not shown), making it the focus of further investigation.
Next, the activity of CpdLC-6888 against diverse C. albicans clinical isolates was evaluated. CaCi-2 and CaCi-17 are isolates obtained from an HIV patient undergoing azole treatment, with CaCi-2 representing an azole-tolerant strain collected early during treatment and CaCi-17 an azole-resistant strain isolated later in treatment (6,7). Specifically, CaCi-17 overexpresses a mutant form of Erg11 that contains a homozygous R467K substitution as well as overexpresses ABC efflux pumps due to a heterozygous substitution in the transcriptional activator Tac1 (6, 7). F5 and G5 represent clinical isolates that overexpress the major facilitator transporter Mdr1 due to substitutions in the transcriptional activator Mrr1 (8,9). In contrast, DPL15 is a strain harboring mutations in the echinocandin drug target gene FKS1 (10) (Fig. 1D). CpdLC-6888 showed potency against DPL15 equivalent to that of the laboratory screening strain CaSS1. In contrast, CaCi-2 and CaCi-17 demonstrated increasing resistance to CpdLC-6888, and F5 and G5 were resistant to all concentrations tested (Fig. 1D). Notably, the resistance observed with CaCi-17 was at least in part due to increased expression of ABC transporters, as laboratory-derived strains that specifically harbored substitutions in Tac1 were also resistant to CpdLC-6888 (11) (Fig. 1D). To further assess CpdLC-6888's spectrum of activity, dose-response assays were conducted against a panel of pathogenic yeasts, including non-albicans Candida species and the basidiomycete Cryptococcus neoformans (Fig. 1E). CpdLC-6888 exhibited potent activity against C. albicans and its closest relative, Candida dubliniensis, moderate activity against the next closest relative, Candida tropicalis, and minimal to no activity against the more divergent species (Fig. 1E). Finally, to further characterize the bioactivity of CpdLC-6888, we assessed its ability to block the C. albicans morphogenetic transition between yeast and filamentous states, a critical virulence trait (12,13). C. albicans was incubated with 2.5 mM CpdLC-6888 overnight before being exposed to 10% heat-inactivated fetal bovine serum (HI FBS) at 37°C for 4 h in the presence or absence of 2.5 mM CpdLC-6888. This CaSS1 for each compound screened at 10 mM in YPD at 30°C for 24 h. Hit compounds, shown in red, were defined as conferring .80% growth inhibition. (B) Chemical structures of the four hit compounds. Note for CpdLC-2800, the E isomer (as shown) was the compound screened; however, the E/Z racemic mixture was used for validation in panel C. (C) Potency of hit compounds was assessed using a doseresponse assay. C. albicans CaSS1 was grown in a 2-fold dilution series of the indicated compound in YPD at 30°C for 24 h. Growth was assessed by measuring optical density at 600 nm (OD 600 ). Values were normalized relative to the respective no-drug control well (see color bar). (D) Dose-response assays with CpdLC-6888 performed as described for panel C for the C. albicans screening strain, CaSS1, the azoletolerant and -resistant C. albicans clinical isolates, CaCi-2 and CaCi-17, the clinical isolates F5 and G5, and the echinocandin-resistant isolate DPL15. The MRR1 allele in F5 contains a mutation that leads to a P683S substitution, and the MRR1 allele in G5 contains a mutation that (Continued on next page) A 2,5-Disubstituted Pyridine Targets C. albicans Erg11 mSphere concentration of compound was selected as it had minimal impact on growth under these conditions. While C. albicans filamented in the presence of serum alone, the addition of CpdLC-6888 blocked filamentation, akin to the noninducing condition (Fig. 1F). Therefore, our chemical screen identified a molecule with novel antifungal activity against C. albicans, which was also able to impede hyphal morphogenesis at subinhibitory concentrations.
CpdLC-6888 putatively inhibits ergosterol biosynthesis through targeting of Erg11. To gain insights into the putative target(s) of CpdLC-6888, haploinsufficiency profiling (HIP) was performed (Text S1). HIP operates under the principle that deletion of one copy of a compound's target gene, or genes involved in related pathways, in a diploid organism will confer hypersensitivity to the compound (14,15). The relative growth of each C. albicans strain in a library of double-barcoded heterozygous deletion mutants covering ;90% of the genome was assessed in the presence of CpdLC-6888 compared to a solvent control (14). Strain-specific molecular barcodes were sequenced, and strains carrying barcodes with a solvent/drug log 2 ratio of reads greater than 5.5 median absolute deviations (MADs) above the median of the entire pool were scored as significantly hypersensitive. Interestingly, the heterozygous deletion mutant for ERG11, the target of the azoles, emerged as the only significant hit identified from analysis of both barcodes ( Fig. 2A).
Erg11 was further explored as the putative target of CpdLC-6888 by examining changes in susceptibility when ERG11 expression was altered. First, the effect of gene repression was assessed with a tetracycline-repressible C. albicans strain in which addition of doxycycline (DOX) repressed ERG11 expression (see Fig. S1 in the supplemental material) (16). Dose-response assays with and without DOX against CpdLC-6888 or the widely deployed azole fluconazole revealed that repression of ERG11 conferred hypersensitivity to both CpdLC-6888 and fluconazole (Fig. 2B). Second, the effect of overexpression of ERG11 was examined using strains with a heterozygous and homozygous gain-of-function mutation in the ERG11 transcriptional regulator UPC2 (Fig. S1). The mutation conferred increased levels of resistance to CpdLC-6888 in a manner similar to that of fluconazole, with the highest resistance seen in the homozygous mutant that also showed the highest expression of ERG11 (Fig. 2C and Fig. S1).
Resistance to azoles is also achieved by loss-of-function mutations in ERG3, a gene that encodes an enzyme in the ergosterol biosynthesis pathway responsible for producing an aberrant sterol intermediate during azole exposure (4). There was a significant increase in resistance to both CpdLC-6888 and fluconazole upon homozygous deletion of ERG3 (Fig. 2C), consistent with the model that CpdLC-6888 inhibits Erg11. These findings are further supported by the lack of activity of CpdLC-6888 seen in the azole-resistant clinical isolate CaCi-17 (Fig. 1D).
The results thus far support CpdLC-6888 as targeting Erg11 in an azole-like manner, despite the fact that it does not possess the canonical five-membered nitrogen-containing azole ring. A well-established feature of the azoles is their synergistic interaction with inhibitors of the molecular chaperone Hsp90, such as geldanamycin. Further, combination treatment results in fungicidal activity (17,18). To determine if CpdLC-6888 acts in a similar manner, dose-response matrices were performed with CpdLC-6888 and geldanamycin. Compound interaction was quantified using the fractional inhibitory concentration index 90 (FICI 90 ), where synergy is represented by a value lower than 0.5 (19). These results indicated that CpdLC-6888 synergizes with geldanamycin, with an FICI 90 value of 0.094 (Fig. 2D, top). Spotting assays in which compound-treated cells were transferred onto drug-free medium were used to assess the cidality of this chemical interaction. Both fluconazole and CpdLC-6888 were found to be fungistatic Images were taken by differential interference contrast microscopy at 40X magnification. alone at concentrations that inhibited growth but became fungicidal when combined with geldanamycin (Fig. 2D, bottom). To investigate potential interactions between CpdLC-6888 and Erg11, computational docking using Rosetta was employed to model binding poses compared to fluconazole (Text S1). Docking of fluconazole returned the expected metal coordination bond between the azole ring nitrogen and heme iron (Fig. 2E, top) (20,21). However, given the absence of the azole ring in CpdLC-6888, the same metal coordination bond was not identified by docking analysis (Fig. 2E, bottom). Instead, docking analysis implicated four candidate residues in the interaction between Erg11 and CpdLC-6888: Y94, L97, F204, and T287. CpdLC-6888 and itraconazole both interact with Y94, while CpdLC-6888 and fluconazole both interact with T287. Thus, this analysis predicts similar amino acids are required for CpdLC-6888 to bind to Erg11 compared to azoles, despite a vastly different compound structure. A close examination of the binding mode unveiled it is the adamantyl group of CpdLC-6888 that interacts with L97 and F204, which form a hydrophobic patch of the binding pocket, interactions that do not occur with azoles. Additional analysis comparing Erg11 sequence within the binding pocket residues across fungal pathogens revealed four residues that are not conserved across the protein in diverse Candida species (Fig. 2F). Interestingly, these residues are found at the opening of the drug-protein binding pocket based on the existing Erg11 structure, and conservation of these residues approximately correlates with the biological activity observed in Fig. 1E.
Finally, to specifically probe Erg11 inhibition, the abundances of the Erg11 precursor lanosterol and the pathway's end product, ergosterol, were quantified through sterol profiling. C. albicans was treated with equivalent inhibitory concentrations of CpdLC-6888 or fluconazole, and sterol abundance was evaluated by LC-MS. Consistent with fluconazole, CpdLC-6888 treatment resulted in a significant increase in lanosterol and a significant decrease in ergosterol relative to solvent-treated controls (Fig. 2G). Collectively, these data provide evidence that CpdLC-6888 targets Erg11 in C. albicans. These findings are intriguing, because while the azoles are established inhibitors of Erg11, CpdLC-6888 appears to share the same target despite lacking the canonical azole ring (4,22). This scaffold, therefore, expands the repertoire of chemical matter capable of inhibiting Erg11 (23). In the current climate where rapidly progressing antifungal resistance paired with a dearth of antifungal treatment options threatens our health care system, CpdLC-6888 represents a new scaffold to explore in an effort to expand our antifungal arsenal. FIG 2 Legend (Continued) assessed by high-throughput sequencing to quantify the abundance of two strain-specific barcodes (UP-TAG and DN-TAG). Strains were considered significantly reduced in abundance if the solvent/drug log 2 ratio was greater than 5.5 median absolute deviations (MADs) above the median for both the UP-TAG and DOWN-TAG or if either the UP-TAG or DOWN-TAG was .5.5 MAD and the opposing TAG was omitted due to low total reads. Significantly enriched UP-TAGs and DOWN-TAGs are shown in blue and red, respectively. Gray dots represent strains that were not classified as hits because neither tag was significant or only one tag was significant, but the other had reads above threshold. (B) CpdLC-6888 and fluconazole dose-response assays performed as described for Fig. 1C with a slight modification. For the parental CaSS1 (ERG11/ERG11) and tetO-ERG11/erg11D C. albicans strains, cultures were grown overnight in YPD followed by an additional night in YPD supplemented with 0.01 mg/mL doxycycline (DOX) to repress ERG11 expression. Strains were then inoculated into assay plates with or without DOX, fluconazole, and CpdLC-6888 as described for Fig.  1C. See color bar. (C) CpdLC-6888 and fluconazole dose-response assays performed as described for Fig. 1C with either the parental SC5314 (UPC2/UPC2) and UPC2 G648D mutant C. albicans strains (top) or with the parental SN95 (ERG3/ERG3) and erg3D/erg3D strains (bottom). See color bar in panel B. (D, top) Dose-response matrices with C. albicans CaCi-2 comparing CpdLC-6888 or fluconazole in combination with geldanamycin. Growth conditions and data analysis were as described for Fig. 1C. See color bar in panel B. Spotting assays (bottom) were performed by removing 2.5 mL of cells from the dose-response matrices after 48 h of growth and spotting onto drug-free YPD agar. Plates were incubated at 30°C for 24 h before being photographed. FICI 90 values are denoted in the upper right. (E) Predicted binding poses from docking Erg11 and heme (green structure) with fluconazole (pink structure, top) and CpdLC-6888 (light blue structure, middle). Within the structures, nitrogen (dark blue), iron (orange), oxygen (red), and fluorine (pale blue) are also indicated. Interacting residues between Erg11 and the molecules are listed in the table and indicated on the structures (yellow) along with the metal coordination bond (black). The hydrophobic patch is indicated by the electrostatic surface. (F) Table listing Erg11 residues present at indicated position from each fungal species as well as overall protein similarity. (G) The abundance of ergosterol (pink) and lanosterol (blue) was quantified by LC-MS in C. albicans SN95 cells treated with solvent, CpdLC-6888, or fluconazole. Values are relative to an internal cholesterol standard, and the fold change relative to the untreated sample is indicated in each respective bar. Errors represent means 6 SD from technical triplicates, and significance was determined by a Welch's two-sided unpaired t test comparing untreated to each treated condition. **, P , 0.01; ***, P , 0.001.