[Helminth derived Immunomodulatory Glycan LNFP3 Impairs Pathogenesis of Peripheral Neuropathic Pain and Spinal Glial Activation].

OBJECTIVES: To investigate the effect of helminth-derived immunomodulatory glycan lacto-N-fucopentaose3(LNFP3) on the pathogenesis of neuropathic pain and spinal glial activation in the corresponding time windows after adult rat tibial nerve permanent transection (modified spared nerve injury, mSNI). METHODS: Ten weeks old male adult Sprague-Dawley (SD) rats weighing 250-300 g were randomly grouped into four groups: sham-operated group (n =6), mSNI group (n =6), mSNI plus bovine serum albumin (BSA) group (n =12) and mSNI plus LNFP3 group (n=12). Rats were subjected to surgical operation or sham operation on the right tibial nerves and were intraperitoneal injected BSA or LNEP3-BSA conjugates by the group design. Animals from each group (n=6 per group) were subjected to the plantar test,von Frey hairs test, pinprick test and acetone test for critical evaluation of region-specific pain responses on the plantar sural and saphenous skin territories of ipsilateral and contralateral hindpaws after injuries. Transverse frozen sections of L3-4 spinal cords from the remaining animals of mSNI plus BSA group and mSNI plus LNFP3 group 7 and 14 d after injury (n=3 for each time point per group)were prepared and subjected to immunofluorescent staining of microglia/macrophage marker [cluster of differentiation molecule 11b (CD11b)] and astrocyte marker [glial fibrillary acidic protein (GFAP)], for analysis of spinal glial activation. RESULTS: After adult rat mSNI, early systematic administration of LNFP3 significantly but not completely attenuated region-specific pathological pain evoked by mechanical and thermal stimuli on the sural and saphenous skin territories of rat hindpaw plantar surfaces in acute (4/5 d after injuries) and subacute (7/8 d and 14/15 d after injuries) phases. Meanwhile, in the ipsilateral spinal cord dorsal horns, this early systematic treatment inhibited microglia/macrophage activation 7 d after injury and astrocyte activation 7 and 14 d after injury. CONCLUSIONS: Early systematic administration of LNFP3 impairs the pathogenesis (acute induction and chronic transition) of neuropathic pain and spinal glial activation in the corresponding time windows after adult rat mSNI.

[Early Systemic Administration of IL-10 Inhibits Neuropathic Pain in Adult Rats with Tibial Nerve Injury].

OBJECTIVES: To determine the effect of early systemic administration of IL-10 on peripheral neuropathic pain induced by tibial nerve permanent transection [modified spared nerve injury (mSNI)]in adult rats. METHODS: Male adult Sprague-Dawley (SD) rats (ten-week old, 250-300 g) with mSNI were randomly divided into mSNI, sham-operated, IL-10 intervention (intraperitoneal injection), PBS intervention (intraperitoneal injection) groups, each containing six rats. Intraperitoneally injections (IL-10 or PBS) were given immediately after surgeries for a single regime with a dosage of 500 uL (0.1 mg/mL). Plantar test, von Frey hairs test, pinprick test and acetone test were performed before and after tibial nerve injuries (0 d, 4/5 d, 7/8 d, 14/15 d) to evaluate region-specific pain responses of the rats on the plantar sural and saphenous skin territories of ipsilateral and contralateral hindpaws. The hindpaw position (on 8 d) of six additional rats with standard SNI was compared with those with mSNI. RESULTS: The rats with standard SNI showed an eversion posture of hindpaws, more prominent than those with mSNI. Region-specific pathological pain evoked by mechanical and thermal stimuli on the sural and saphenous skin territories of the plantar surfaces of rat hindpaws was demonstrated on the ipsilateral rather than contralateral hindpaws. This effect was shown in the rats with mSNI but not in those with sham operations. Compared with PBS, early intraperitoneal injection of IL-10 significantly and persistently attenuated either allodynia or hyperalgesia in the rats with mSNI. CONCLUSIONS: Tibial nerve permanent transection models of adult rats can be used as a simple but useful rodent model of peripheral neuropathic pain. Early systemic administration of IL-10 impairs the pathogenesis of neuropathic pain induced by tibial nerve injuries.

[Senescence-associated Beta Galactosidase Expression in Rat Schwann Cells after Chronic Denervation].

OBJECTIVE: To investigate the effect of prolonged axon depletion on senescence-associated beta galactosidase (SA-ß-gal) expression in Schwann cells (SCs) of adult rats. METHODS: Male adult Sprague-Dawley (SD) rats were randomize grouped into sham-operated group and denervation groups for 1 week, 2 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks. Rats were subjected to right sciatic nerve transection. After particular denervation duration for the distal stumps, animals were anesthetized and perfused. Proximal stumps of 5 mm and distal stumps of 10 mm from injured nerves, and the corresponding segments from the sham groups and contralateral nerves were harvested and prepared for SA-ß-gal staining to detect SA-ß-gal expression. Then, additional injured distal stumps denervated for 8 weeks were employed for determining cellular distribution of SA-ß-gal expression by co-labeling of SA-ß-gal and SC-specific protein (S100ß). RESULTS: SA-ß-gal expression transiently increased in distal tips of proximal stumps 2 weeks after adult rat sciatic nerve transection without suture. In contrast, in the distal stumps of transected adult rat sciatic nerves, axon depletion for 2 weeks increased SA-ß-gal expression, and the increased expression of SA-ß-gal remained constant after prolonged denervation durations. Furthermore, combination of SA-ß-gal staining with S100ß immunofluorescence staining showed that SA-ß-gal expression. was exclusively present in denervated SCs. CONCLUSION: Prolonged axon depletion increased SA-ß-gal expression in adult rat SCs.

Behavioral characterization of neuropathic pain on the glabrous skin areas reinnervated solely by axotomy-regenerative axons after adult rat sciatic nerve crush.

In cranial and spinal nerve ganglia, both axotomized primary sensory neurons without regeneration (axotomy-nonregenerative neurons) and spared intact primary sensory neurons adjacent to axotomized neurons (axotomy-spared neurons) have been definitely shown to participate in pain transmission in peripheral neuropathic pain states. However, whether axotomized primary sensory neurons with regeneration (axotomy-regenerative neurons) would be integral components of neural circuits underlying peripheral neuropathic pain states remains controversial. In the present study, we utilized an adult rat sciatic nerve crush model to systematically analyze pain behaviors on the glabrous plantar surface of the hindpaw sural nerve skin territories. To the best of our knowledge, our results for the first time showed that heat hyperalgesia, cold allodynia, mechanical allodynia, and mechanical hyperalgesia emerged and persisted on the glabrous sural nerve skin areas after adult rat sciatic nerve crush. Interestingly, mechanical hyperalgesia was sexually dimorphic. Moreover, with our optimized immunofluorescence staining protocol of free-floating thick skin sections for wide-field epifluorescence microscopic imaging, changes in purely regenerative reinnervation on the same skin areas by axotomized primary sensory afferents were shown to be paralleled by those pathological pain behaviors. To our surprise, Protein Gene Product 9.5-immunoreactive nerve fibers with regular and large varicosities ectopically emigrated into the upper dermis of the glabrous sural nerve skin territories after adult rat sciatic nerve crush. Our results indicated that axotomy-regenerative primary sensory neurons could be critical elements in neural circuits underlying peripheral neuropathic pain states. Besides, our results implied that peripheral neuropathic pain transmitted by axotomy-regenerative primary sensory neurons alone might be a new dimension in the clinical therapy of peripheral nerve trauma beyond regeneration.

ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.

Differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush.

Peripheral nerve functional recovery after injuries relies on both axon regeneration and remyelination. Both axon regeneration and remyelination require intimate interactions between regenerating neurons and their accompanying Schwann cells. Previous studies have shown that motor and sensory neurons are intrinsically different in their regeneration potentials. Moreover, denervated Schwann cells accompanying myelinated motor and sensory axons have distinct gene expression profiles for regeneration-associated growth factors. However, it is unknown whether differential motor and sensory functional recovery exists. If so, the particular one among axon regeneration and remyelination responsible for this difference remains unclear. Here, we aimed to establish an adult rat sciatic nerve crush model with the nonserrated microneedle holders and measured rat motor and sensory functions during regeneration. Furthermore, axon regeneration and remyelination was evaluated by morphometric analysis of electron microscopic images on the basis of nerve fiber classification. Our results showed that Aα fiber-mediated motor function was successfully recovered in both male and female rats. Aδ fiber-mediated sensory function was partially restored in male rats, but completely recovered in female littermates. For both male and female rats, the numbers of regenerated motor and sensory axons were quite comparable. However, remyelination was diverse among myelinated motor and sensory nerve fibers. In detail, Aß and Aδ fibers incompletely remyelinated in male, but not female rats, whereas Aα fibers fully remyelinated in both sexes. Our result indicated that differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush.