Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A

Post-SCI Hypersensitivity to Mechanical Stimuli.

To assess the potential therapeutic effect of HUP-A, we first used our clinically relevant model of moderate, static-compression SCI (Fig. 1E) to profile reproducible post-SCI mechanical hypersensitivity in both the fore- and hindpaws of experimental rats. In a modified von Frey filament assay (Fig. 2A), the pre-SCI paw-withdrawal sensitivity threshold was 0.63 ± 0.06 g for the forepaws and 2.00 ± 0.33 g for the hindpaws (n = 4; Fig. 2B). In the acute phase after SCI (i.e., 24 h after lesion, without treatment), the force required to trigger a withdrawal response increased drastically, to >3 g for the forepaws and >50 g for the hindpaws (day 1 after SCI, Fig. 2B). The lack of response in the acute phase is attributable to “spinal shock” associated with the transient shutdown of sensorimotor function in the spinal cord acutely after SCI (24). In the subacute and chronic phases after injury, the withdrawal threshold decreased gradually to below the pre-SCI level in the forepaws (0.11 ± 0.02 g in week 2, P < 0.01, and 0.15 ± 0.01 g in week 3, P < 0.03; n = 4). The hindpaws took a week longer to develop hypersensitivity (1.97 ± 1.17 g in week 2, P > 0.05, and 0.47 ± 0.10 g in week 3, P < 0.05; n = 4); the results in both paws indicate the development of persistent hypersensitivity to mechanical stimuli (Fig. 2B).

Fig. 2.

(A) The post-SCI chronic development of above-injury-level and below-injury-level hypersensitivity was evaluated with a modified von Frey filament test, as diagrammed. The forepaw- and hindpaw-withdrawal thresholds to von Frey filaments of several stiffnesses were tested in acclimated and resting conscious rats. The paw area probed by the filament is shaded in gray and indicated by the long arrow. (B) Baseline data of changes in the paw-withdrawal threshold over the study’s time course. (C and D) The effect is dose dependent. I.p. injection of varying doses of HUP-A resulted in reproducible anti-hypersensitivity effects, i.e., increased withdrawal thresholds of the forepaws (C) and hindpaws (D), in the chronic stage of SCI (i.e., 4 wk after injury). Sal, saline.

To evaluate pharmacological specificity and potential side effects of systemic HUP-A administration, following a sequential design with adequate drug washout intervals (i.e., >5× t_1/2), we injected three doses of HUP-A i.p. following preinjection of an equivolume of saline in the same rats as self-control in week 4 after SCI (10). HUP-A (500, 167, 50 µg/kg, i.p.; n = 4 rats per group) induced dose-dependent anti-hypersensitivity (Fig. 2 C and D). Additionally, after drug administration fine muscle tremors, primarily in the forepaw digits, were observed, indicating systemic HUP-A–mediated AChE inhibition at the neuromuscular junction (10, 12); however, we did not observe any worsening of the hindlimb muscle spasticity that typically develops 3–4 wk after lower thoracic SCI (24). This sign persisted for ∼0.2–2 h in the group with the highest dose of HUP-A. In contrast, the analgesic effects of HUP-A lasted much longer, averaging up to 6 h with bolus i.p. delivery of HUP-A (500 µg/kg; Fig. 2 C and D). We observed no other isolated or cumulative adverse effects and particularly no effects representing gastrointestinal hyperactivity (23).

Continuous Intrathecal Administration of HUP-A Induces Sustainable Suppression of Post-SCI Hypersensitivity.

We next tested a continuous drug-delivery regimen with a s.c.-implanted osmotic pump that delivered HUP-A directly and continuously into the intrathecal (i.t.) space at L5–L6 for 2 wk (0.5 μL/h × 24 h × 14 d; total dose: 90 µg/168 µL) (Fig. 3A), with the aim of maintaining the long-term effect of central anti-hypersensitivity and preventing systemic side effects (20). Continuous i.t. delivery of HUP-A via the osmotic pump effectively eliminated the forepaw and hindpaw hypersensitivity to mechanical stimuli. A strong analgesic effect was noted between weeks 1 and 3 after pump implantation: The post–HUP-A treatment sensitivity threshold was significantly higher (Fig. 3 E–H) (n = 7 per group) than the pretreatment/post-SCI baseline sensitivity thresholds of 0.60 ± 0.04 g and 2.00 ± 0.18 g for the fore- and hindpaws, respectively (Fig. 3 C and D). The anti-hypersensitivity and analgesic effects were HUP-A dependent, because the effect was first spotted ∼24 h after pump implantation (Fig. 3 E–H), reflecting the time needed for HUP-A to course through the microtubing before entering the i.t. space (additional details are given in Materials and Methods), and a return of hypersensitivity was observed 2 wk after depletion of HUP-A from the osmotic pump (i.e., 4 wk after s.c. pump insertion). However, readministration of the same dose reestablished the anti-hypersensitivity effect of HUP-A without apparent tolerance or adverse effects (Fig. 3 E and F; note: the effectiveness of the second HUP-A pumping also lasted for ∼3 wk). To check whether HUP-A’s augmentation of central muscarinic activation of cholinergic transmission indeed played a definitive role in managing hypersensitivity, we administered i.t. bolus doses (15 µg/10 µL) of atropine, a nonselective muscarinic cholinergic antagonist (Fig. 3B), into SCI rats 20 d after onset of HUP-A infusion (i.e., 5 d after the exhaustion of the contents of the HUP-A pump). The timing of this test allowed a cleaner assessment of HUP-A’s anti-hypersensitivity capability derived from cholinergic activation per se, because the anti-central pain effect of HUP-A mediated by NMDA antagonism diminishes swiftly after the end of drug administration (i.e., when HUP-A infusion became exhausted 2 wk after pump insertion) (20, 21). Injection of atropine indeed abolished HUP-A–mediated anti-hypersensitivity immediately, indicating that the continued analgesic effect observed in week 3 after minipump implantation resulted largely from cholinergic agonism via HUP-A’s longer-lasting inhibition of AChE ( Fig. 3 E–H, Right) (20). Interestingly, although the observed increase in paw-withdrawal thresholds in response to mechanical stimuli is consistent with the observed effects on nonnoxious paw pressure and nociceptive pinch reflexes during the second week of HUP-A infusion (Fig. 3 D, G, and H), open-field locomotor performance [measured by the Basso–Beattie–Bresnahan (BBB) score; Fig. 3I] and coordinated body posture control (measured by the inclined plane angle threshold for maintaining head-downward posture; Fig. 3J) were not affected significantly by i.t. administration of HUP-A.

Fig. 3.

By using an osmotic pump-mediated i.t. delivery, we provided more targeted administration of HUP-A. (A) Schematic representation of the s.c.-implanted osmotic pump for i.t. delivery. (B) Schematic representation of i.t. injection of atropine. (C) Baseline data before and after SCI: changes in paw-withdrawal threshold in response to a modified von Frey filament test. (D) Baseline data before and after SCI: changes in paw-withdrawal threshold in response to conventional pressure and painful spinal reflex tests. (E–H) In E and F, the forepaws and hindpaws demonstrated durable (i.e., up to 3 wk after HUP minipump implantation) increases in the sensory threshold to mechanical stimuli. (E–H, Far Right) An i.t. bolus injection of atropine (B) swiftly and temporarily blocked the anti-hypersensitivity effect of HUP-A. In G and H, hind-limb sensory reflexes support the findings obtained for the paw-withdrawal threshold. However, I and J indicate that HUP treatment did not improve locomotion as evaluated with the open-field locomotion test (BBB score) and the head-downward inclined plane test (24).

Histopathology and Immunocytochemical Analyses of the Spinal Cords.

Although gross pathology at the lesion epicenter assessed by solvent blue and hematoxylin staining (Fig. 4A) did not demonstrate significant differences in spared tissue volume (Fig. 4B) or in the number of surviving motor neurons (Fig. 4C), further immunocytochemical (ICC) analyses revealed that specific neuroinflammation markers were differentially expressed in the i.t. HUP-A–treated spinal cords and saline-treated controls, especially in the cervical and lumbar regions that receive sensory inputs from the fore- and hindpaws, respectively (25–27). Expression of GFAP, a classic marker of astrocytes whose expression is increased markedly in reactive gliosis and neuroinflammation (26, 27), was observed in all chronically injured spinal cords. However, rats treated with i.t. HUP-A demonstrated significantly reduced GFAP immunoreactivity in cervical and lumbar spinal cord regions (Fig. 5A), as well as a significantly lower immunoreactivity level for CD68, a marker for activated microglia and macrophages, in the lumbar spinal cord (26, 27); no effect of HUP-A on cervical CD68 immunoreactivity was detected (Fig. 5B). Immunoreactivity for the inducible form of nitric oxide synthase (iNOS), a critical neuroinflammatory mediator expressed by immune cells and microglia (27), was significantly lower in the cervical (Fig. 5C, red immunoreactivity) and lumbar (Fig. 5D) spinal cords of the HUP-A–treated group than in the saline-treated controls. Immunoreactivity for CD86, which is expressed by M1 macrophages that have a detrimental role after SCI (28), was reduced significantly in cervical (Fig. 5E, green cells) and lumbar (Fig. 5F) regions of the HUP-A–treated group compared with controls, whereas immunoreactivity for arginase1, which is expressed by M2 macrophages that have a beneficial role after SCI (28), was comparable in the cervical and lumbar spinal cord in both groups (Fig. 5 E and F, red cells). The differences in the immunoreactivity profiles of cervical CD68, iNOS, and CD86 may carry further mechanistic information and should be investigated systematically in future studies (29).

Fig. 4.

Lesion epicenter histopathology (S/H staining) (A) was similar in the HUP-A–treated and saline-treated control (CTL) spinal cords based on analysis of the spared tissue volume in the epicenter (B) and the number of surviving motoneurons in the region surrounding the epicenter. Motor neuron numbers (mean ± S.E.) were calculated by summing motor neuron numbers of 6 coronal sections (20 μm) selected from each mm of tissue rostral and caudral to the epicenter (i.e., -3 – 3 mm) and then averaged (n = 7/group) (C). CTL, control. (Magnification: 5×).

Fig. 5.

Neuroinflammation and reactive astrogliosis developed in the cervical and lumbar spinal cord (i.e., regions rostral and caudal to the lesion site, respectively) as evidenced by strong expression of GFAP (A); activation of microglia (CD68 is a marker for activated microglia and macrophages) (B); expression of iNOS (C and D); and invasion of M1 macrophages (E and F) as determined by CD86 staining. HUP-A administration significantly ameliorated all indices of neuroinflammation. However, no changes were observed for immunoreactivity scales of the beneficial M2 macrophages (Arginase, Argi stain). (G–J) HUP-A administration protected white matter (WM) tissue, i.e., mitigating the chronic loss of WM myelin (assessed by immunocytochemistry against oligodendrocytic markers, MBP and CNPase), evident in the control group. Chronic HUP-A administration significantly preserved the myelin in the WM compared with the CTL group. (K and L) Comparison of expression levels immunoreactivity (IR) of neurofilaments M (NFM: (K) and H (NFH) (L) in transverse epicenter sections from the HUP-A–treated and control (CTL) groups (i.t.; n = 7 per group). Because there is no statistical difference between the two groups in the average total pixel numbers per section (768,908 ± 96,330 vs. 752,678 ± 67,401 pixels; n = 3 sections per rat; n = 7 rats per group; P > 0.05, Student t test), the percentages of NFM ⁽K, red) or NFH ⁽L, green) pixels within the total image field of the white matter were measured and compared. The result suggests that there is no significant difference in neurite presence or regeneration in the spared tissue around injury epicenters. *P < 0.05, Student t test. (Scale bars: 100 μm.)

The immunoreactivity of the oligodendrocyte markers CNPase (Fig. 5 G and H) and myelin basic protein (MBP) (Fig. 5 I and J) demonstrated significantly increased preservation of myelin in the lumbar white matter of HUP-A–treated rats, suggesting that i.t. HUP-A treatment did not trigger myelin toxicity, as has been reported for some prototype NMDA antagonists (30). Instead, HUP-A protected against demyelination in chronic SCI. However, immunoreactivity levels of CNPase, a marker of immature oligodendrocytes, were comparable in the cervical spinal cords of the HUP-A– and saline-treated groups (Fig. 5H), possibly because immature oligodendrocytes are less involved in chronic demyelination after SCI. Moreover, we did not find a significant difference in immunoreactivity levels of neurofilament M or H in epicenter tissue sections from HUP-A–treated and control rats (Fig. 5 K and L). These findings collectively support the hypothesis that chronic administration of HUP-A does not introduce further toxicity in the injured spinal cord and that HUP-A treatment, although not triggering axonal regeneration (Fig. 5 K and L), protects myelin against the chronic damage resulting from neuroinflammation that is mediated largely by locally activated microglia and astroglia and by macrophage invasion (25–29).

To explore further the mechanistic basis for the observed benefits of the HUP-A treatment, we evaluated the immunoreactivity levels for the GluR2 (also called “GluR-B”) subunit of the AMPA receptor, which is Ca ^{impermeable and is in balance with numbers of Ca-permeable GluR1 subunits to regulate Ca permeability in DH neurons where nociceptive relaying/processing occurs (}31). Average GluR2 immunoreactivity was significantly higher in the cervical and lumbar spinal cords of the HUP-A–treated group (Fig. 6 A and B), suggesting that HUP-A–activated cholinergic interneurons might have helped reduce the post-SCI loss of GluR2-expressing neurons that is partially responsible for the development of hypersensitivity (Discussion and refs. 31 and 32). More support for this notion is derived from our immunoreactivity data on the nociceptive mediators neurokinin-1 receptor (NK1R) (green immunoreactivity, Fig. 6 C and D) and calcitonin receptor-like receptor (CRLR) (red immunoreactivity, Fig. 6 C and D). Both markers were decreased significantly in the cervical and lumbar DH spinal cord of the HUP-A–treated rats. Additionally, immunoreactivity for substance P, a nociceptive neurotransmitter, was decreased significantly in cervical and lumbar spinal cord DH regions of the HUP-A–treated rats. Immunoreactivity for the immediate early gene (IEG) cFos, which is increased in neurons with enhanced activity and is used frequently as a marker of neuronal activation in neuropathic pain, was decreased significantly in laminae I–III of the cervical and lumbar DH spinal cord in the HUP-A–treated group (Fig. 6 G and H). Similarly, Homer1a, an activity-dependent inducible dominant-negative form of the linking protein that can disrupt the mGluR-signaling complex and regulate nociceptive plasticity at spinal synapses (33), was significantly lower in the cervical and lumbar DH of HUP-A–treated rats than in controls (Fig. 6 I and J). This result suggests that, with HUP-A treatment, DH sensory neurons became less activated by nociceptive signaling. Additionally, group average immunoreactivity intensity of the neurotrophin receptor p75NTR also was significantly weaker in the cervical and lumbar DH of the HUP-A–treated rats (Fig. 6 K and L) than in the control group, indicating decreased local sensory neurite sprouting. This decrease also may contribute to the observed anti-hyperalgesic effect of HUP-A, because aberrant neurite sprouting in the spinal cord contributes to the onset and maintenance of neuropathic pain in other clinical settings such as postherpetic or postcrush neuralgia (34).

Fig. 6.

HUP-A administration significantly inhibited nociceptive signaling in the cervical and lumbar spinal cord, albeit to a lesser degree in the cervical segment relatively distant from the lesion site. (A and B) Average expression of the GluR2 subunit of the AMPA receptor was higher in HUP-A–treated lumbar and cervical spinal cords. (C and D) CRLR and NK1R receptor expression in the DH was significantly lower in HUP-treated rats. (E and F) Substance P expression in the DH was decreased in HUP-treated rats. (G and H) cFos expression indicates decreased neuronal activity in the dorsal horn of the cervical and lumbar spinal cords of HUP-A-treated rats. (I and J) Average immunoreactive level of Homer1a, an endogenous protein with an important role in synaptic plasticity through an activity-dependent remodeling of the postsynaptic density in DH sensory neurons of the spinal cord, was significantly lower in HUP-A–treated rats. (K and L) Average immunoreactive level of neurotrophin receptor p75NTR also was significantly lower in HUP-A–treated rats, suggesting decreased neurite sprouting that also may contribute to the better-maintained sensory threshold (34). *P < 0.05, Student t test. (Scale bars: 100 μm.)

Surgical Model of Moderate Static Compression SCI.

All procedures were evaluated and approved by the Institutional Animal Care and Use Committee of Harvard Medical School. Young adult female Sprague–Dawley rats (200–235 g body weight, n = 20; Taconic) were anesthetized with ketamine (75 mg/kg i.p.) and xylazine (10 mg/kg i.p.) before operation. Under a surgical stereomicroscope, dorsal laminectomy at thoracic vertebra 10 (T10) was performed to expose the spinal cord. After hemostasis was induced with Gelfoam (Pfizer), the rat body was suspended with a Teng Laboratory spine stabilization frame (fabricated at the Scientific Instrument Shop, Harvard University School of Engineering and Applied Sciences, Cambridge, MA), with 1-cm clearance between the ventral side of the rat and the frame base platform. A 35-g stainless steel impounder was lowered gently upon the dura surface via a micromanipulator, and the entire weight was allowed to compress the cord for 5 min (Fig. 1E). The lesion zone then was closed with sutures and stainless wound clips (Fine Science Tools Inc.), and the rat was allowed to recover in a clean, heated cage. Lactated Ringer’s solution (10 mL/d, s.c.) was given daily for 5 d postoperation, and the urinary bladder was evacuated manually without urinary catheterization twice daily until reflex bladder function became reestablished (24).

HUP-A Preparation and Treatment.

HUP-A [5R(5a,9b,11E)]-5-amino11ethlidene-5,6,9,10-tetrahydro-7-methyl-5,9-methanocycloocta[b]pyridin-2(1H)-one (molecular weight: 242.32; HPLC/UV purity: 98%; specific rotation: −152.8°; appearance: white powder] (Fig. 1A) was purchased from ChromaDex. Three weeks after SCI, HUP-A was administered as follows: (i) for the i.p. study (bolus dose treatment, n = 4): After sterile saline (2 mL per rat, i.p.) administration together with subsequent behavioral evaluation to obtain vehicle-control outcome measures, HUP-A, dissolved in 10% beta cyclodextrin in sterile saline (Sigma-Aldrich) at a concentration of 10 µg/µL before saline dilution to the final dose of 500, 167, or 50 µg/kg in a 2-mL bolus volume per rat for three treated groups, respectively (20), was administered. The injection was done with syringe needle passing 5 mm upward in the subdermal layer before penetrating through the muscle and peritoneal membrane layer to prevent postinjection leak and a brief withdrawal of the plunger to ensure no blood vessel piercing. After each dosing of saline followed with HUP-A (i.e., 500, 167, and 50 µg/kg with intervals of 24 h and 48 h, respectively), tests were performed using standard behavior batteries (see below). (ii) Implantation of the i.t. osmotic pump for chronic 2-wk treatment: HUP-A dissolved in 10% beta cyclodextrin in sterile saline (Sigma-Aldrich) was loaded in Alzet osmotic pumps (Durect Corporation) at a final dose per pump of 90 µg/168 µL to provide a 14-d i.t. continuous supply (pumping rate: 0.5 μL/h; total dose: 12 µL/d × 14 d = 168 µL). After reanesthetization and a small incision to expose the L5–L6 space, a dura puncture was made by an 18-G needle. A PE20 sterile catheter was inserted through the puncture with the beveled tip facing rostrally and was secured with a suture after a brief sign of tail flick was elicited by a light touch of nerve roots in the cauda equina to confirm proper i.t. penetration. The catheter was prefilled with 12 µL of sterile saline before the insertion and was connected to an osmotic pump filled with either HUP-A (treatment group; n = 7) or sterile saline containing 10% beta cyclodextrin (control group, n = 7). The tubing also was sutured to the fascia and musculature near the connection site with the s.c. embedded pump. The skin incision was closed with stainless steel wound clips, and standard postprocedure care was given (52). It took ∼24 h at a pumping rate of 0.5 μL/h, for HUP-A or saline to start entering the i.t. space. We first validated the effectiveness of the i.t. delivery by microinjection of 10 µL of 0.2% trypan blue via PE20 tubing in a pilot experiment (n = 2). (iii) I.t. redosing and atropine administration: The first round of HUP-A i.t. delivery was followed by a 2-wk “wash-out” period to permit the HUP-A effect to subside. We then started a second cycle of HUP-A treatment by replacing the HUP-A pump. The same sets of behavioral assays were performed, and 20 d later we administered a bolus dose of atropine (15 µg/10 µL, i.t. at L5–L6) to determine the effect of muscarinic antagonism on analgesic impact of HUP-A.

Behavioral Monitoring.

A battery of behavioral tests was performed by observers blinded to the treatments on the day before the surgery, 1 d after SCI, and weekly thereafter to assess functional deficits and nociceptive changes. These tests evaluated open-field locomotion based on the BBB scale, the ability to maintain coordinated posture control on an inclined plane, the contact-righting reflex, the hindpaw-placing reflex, and the withdrawal reflex in response to pinch and pressure. In addition, after 30 min acclimation, the rats were subjected to a paw hyperalgesia test (modified von Frey filament test) to compare the withdrawal threshold before and after SCI or treatments, followed by hourly testing for up to 8 h after i.p. HUP-A injection and for 1 d, 2 d, and weekly thereafter up to 4 wk posttreatment for the i.t. HUP-A delivery study. Using the data obtained, we determined that with the α value (probability of rejecting H₀ when H₀ is true) being set at 5%, an average sample value of 0.41 g (post-SCI baseline sensory threshold), mean test value (posttreatment) of 3.1 g, and group size of four rats, the statistical power (i.e., 1-β) equals ∼100%, indicating that the β value (i.e., the probability of not rejecting H₀ when H₀ is false) = ∼0. The BBB and paw hyperalgesia test data were analyzed by repeated-measures ANOVA and by Bonferroni post hoc tests for multiple group comparisons with Analyze-it Statistics (Analyze-it Software Ltd. on Excel (Microsoft) (24, 52).

Histopathological Analysis.

Rats were killed with an overdose i.p. injection of anesthetics followed by intracardiac perfusion with 4% (wt/vol) paraformaldehyde (PFA) in 0.1 M phosphate buffer at pH 7.4. Brain and spinal cord were dissected carefully, postfixed in 4% (wt/vol) PFA overnight at room temperature, dehydrated in 30% (wt/vol) sucrose at 4 °C for 24 h, and frozen in isopentane (Sigma-Aldrich) at −45 °C. The tissue then was encased in OCT compound (Sakura Finetek) and cryosectioned transversely at 20 µm. Sections representative of each millimeter of the spinal cord at and around the injury epicenter were chosen for solvent blue (a myelin stain, similar to luxol fast blue)/hematoxylin (S/H; both from Sigma) staining for analysis of lesion volume and counting of motor neurons. Lesion volume was measured based on the S/H staining from the area of the visualized lesion region in each representative section per millimeter of tissue and was calculated based on total lesion length. The motor neuron count was carried out as previously described (52). The average results (n = 7 for each study group) were compared with a paired t test performed by the software described above.

ICC Analysis of Signaling Markers Posttreatment.

Representative spinal cord cross-sections were selected from the thoracic region rostral to the SCI epicenter and from cervical and lumbar regions for ICC probing of the post-SCI development of neuroinflammation and mechanistic markers of neuropathic pain pathways involved in post-SCI hyperalgesia. Sections were washed with PBS containing Triton X-100 (0.3%) and incubated in 5% (vol/vol) normal donkey serum. The primary antibodies used are listed in Table 1. Secondary antibodies were from Jackson ImmunoResearch Laboratories Inc. Slides were coverslipped using mounting medium containing DAPI (Vector LabsA) for imaging with an Axiovert200 microscope and digital Axiocam camera (Carl-Zeiss Microimaging) or with a Zeiss LSM1 confocal microscope equipped with Zeiss Zen 2011 software (Carl-Zeiss Microimaging). Semiquantification of positive ICC signals was performed with ImageJ (National Institutes of Health) and Photoshop (Adobe) software. A threshold signal-level range was determined by averaging the pixel brightness of the background and the strongly labeled structures, and the percentage of positively labeled pixels within the visual field was calculated to indicate the overall signal strength in the stained tissue. After confirmation of size compatibility for selected regions between samples, the average pixel numbers of ICC-positive labeling of a specific marker were compared in the treated and control groups (n = 7 per group) with a Student t test.