Evaluation of Vascular Endothelial Growth Factor (VEGF) and Its Family Member Expression After Peripheral Nerve Regeneration and Denervation.

Vascular endothelial growth factor (VEGF) represents one of the main factors involved not only in angiogenesis and vasculogenesis but also in neuritogenesis. VEGF plays its function acting via different receptors: VEGF receptor1 (VEGFR-1), VEGF receptor2 (VEGFR-2), VEGF receptor3 (VEGFR-3), and co-receptors Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2). This study reports on the first in vivo analysis of the expression of VEGF and VEGF family molecules in peripheral nerve degeneration and regeneration: for this purpose, different models of nerve lesion in rat were adopted, the median nerve crush injury and the median nerve transaction followed or not by end-to end microsurgical repair. Results obtained by real time polymerase chain reaction showed that VEGF and VEGF family molecules are differentially expressed under regenerating and degenerating condition, furthermore, in order to study the modulation and involvement of these factors in two different regenerative models, crush injury and end-to-end repair, protein expression analysis was evaluated. In addition, immunohistochemical analysis allowed to state a glial localization of VEGF and VEGFR-2 after peripheral nerve crush injury. Finally in vitro assay on primary Schwann cells culture show that VEGF165 stimulation increases Schwann cells migration, a major process in the promotion of neurite outgrowth. Anat Rec, 301:1646-1656, 2018. © 2018 Wiley Periodicals, Inc.

Changes in GABA and GABA(B) receptor expressions are involved in neuropathy in the rat cuneate nucleus following median nerve transection.

This study examined the relationship between changes in GABA transmission and behavioral abnormalities after median nerve transection. Following unilateral median nerve transection, the percentage of GABA-like immunoreactive neurons in the cuneate nucleus and that of GABA(B) receptor-like immunoreactive neurons in the dorsal root ganglion in the injured side decreased and reached a nadir at 4 weeks after median nerve transection. Four weeks after bilateral median nerve transection and intraperitoneal application with saline, baclofen (2 mg kg⁻¹), or phaclofen (2 mg kg⁻¹) before unilateral electrical stimulation of the injured median nerve, we investigated the level of neuropeptide Y release and c-Fos expression in the stimulated side of the cuneate nucleus. The neuropeptide Y release level and the number of c-Fos-like immunoreactive neurons in the baclofen group were significantly attenuated, whereas those in the phaclofen group had increased compared to the saline group. These findings indicate that median nerve transection reduces GABA transmission, promoting injury-induced neuropeptide Y release and consequently evoking c-Fos expression in cuneate nucleus neurons. Furthermore, this study used the CatWalk method to assess behavioral abnormalities in rats following median nerve transection. These abnormalities were reversed by baclofen treatment. Overall, the results suggest that baclofen treatment block neuropeptide Y release, subsequently lessening c-Fos expression in cuneate neurons and consequently attenuating neuropathic signal transmission to the thalamus.

Expression of antioxidant molecules after peripheral nerve injury and regeneration.

Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of antioxidant molecules that occur after injury and regeneration of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the absence or presence of microsurgical repair. The expression profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymerase-1 (PARP-1). The results showed that, in the proximal nerve stump, MT-3 mRNA expression was significantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed significant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regulated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regulated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these findings indicate that antioxidant molecules are differentially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

Performance of a repetitive task by aged rats leads to median neuropathy and spinal cord inflammation with associated sensorimotor declines.

Epidemiological studies have demonstrated a relationship between advancing age and susceptibility to risk factors for median neuropathies and musculoskeletal disorders. In this study, we determined if performance of a voluntary reaching task by aged rats induced sensorimotor declines, median nerve dysfunction and increased inflammatory cytokines in peripheral nerves, muscle and spinal cord neurons. Aged (14 mon) rats were trained for 15 min/day for 4 weeks to learn a high repetition, low force (HRLF) task (19 reaches/min; 15% maximum pulling force). Aged task rats performed the task for 2 h/day, 3 days/wk, for 12 weeks (until they were 18 mon of age). No behavioral changes were detected in normal controls (NC) or food-restricted controls (FR C) as they aged. However, grip strength declined in HRLF rats in weeks 6-12 (P<0.01 each) and 12-week trained-only rats (TR; P<0.05), compared to NC. Mechanical hypersensitivity was present in weeks 9 and 12 HRLF reach limb forepaws (P<0.01 and P<0.05, respectively), and 12-week HRLF support limb forepaws (P<0.01) and hindpaws (P=0.03), compared to NC. By week 12, median nerve conduction velocity declined 23%, bilaterally, in HRLF (P<0.001 each), and 13% in TR (P<0.05), compared to NC. Tumor necrosis factor alpha (TNFα) increased in 12-week HRLF muscle (P=0.005), median nerve (P<0.01), and neurons in superficial lamina of HRLF cervical spinal cords (P<0.01), compared to NC. interleukin 1 beta (IL1ß) also increased in superficial lamina neurons (P<0.01). Loss of grip strength was correlated with median nerve conduction slowing (r=0.70) as well as increased nerve and muscle TNFα (r=-0.38 and r=-0.41, respectively); decrease in forepaw withdrawal thresholds was correlated with median nerve conduction slowing (r=0.81), increased nerve TNFα (r=-0.59), and increased TNFα and IL1ß in neurons in spinal cord dorsal horns (r=-0.52 and r=-0.47, respectively). Thus, aged rats performing a repetitive task exhibited sensorimotor declines that were associated with decreased median nerve conduction, and increased pro-inflammatory cytokines in the median nerve and cervical spinal cord neurons.

Neuropeptide Y modulates c-Fos protein expression in the cuneate nucleus and contributes to mechanical hypersensitivity following rat median nerve injury.

This study sought to investigate the effects of injury-induced neuropeptide Y (NPY) on c-Fos expression in the cuneate neurons and neuropathic pain after median nerve injury. Four weeks after median nerve transection (MNT), the injured nerves stimulated at low intensity (0.1 mA) expressed significantly less NPY-like immunoreactive (NPY-LI) fibers in the cuneate nucleus (CN) than those stimulated at high intensities (1.0 mA and 10 mA). Conversely, a significantly higher number of c-Fos-LI cells were observed in the CN in rats stimulated with 0.1 mA compared to those stimulated with 1.0 mA or 10 mA. These results suggest that more NPY was released following low-intensity stimulation, and consequently fewer NPY-LI fibers and more c-Fos-LI cells were identified in the CN. Furthermore, the number of c-Fos-LI cells as well as the percentage of c-Fos-LI cuneothalamic projection neurons (CTNs) in the CN was markedly decreased after injection of NPY receptor antagonist along with retrograde tract-tracing method, indicating that NPY regulated c-Fos expression. In rats with median nerve chronic constriction injury (CCI), intracerebroventricular injection of NPY aggravated mechanical allodynia and low-intensity stimulus-evoked c-Fos expression, both of which were reversed by injection of NPY receptor antagonist. However, thermal hyperalgesia was not affected by injection of these two reagents. Taken together, these findings suggest that more NPY release, following low-intensity electrical stimulation of the injured nerve, significantly induces c-Fos expression in the CTNs, which possibly provide the ascending thalamic transmission of neuropathic pain signals.

Localization and changes of intraneural inflammatory cytokines and inducible-nitric oxide induced by mechanical compression.

STUDY DESIGN: Investigation of intraneural inflammation induced by mechanical compression. OBJECTIVES: In order to investigate the mechanism of neuropathy, this study used a median nerve compression model in dogs. Immunohistochemistry was used to examine the localization and changes of inflammatory cytokines and nitric oxide (NO). SUMMARY OF BACKGROUND DATA: The manifestation of pain at sites of inflammation has a close relationship with the release of mediators from macrophages such as interleulin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), as well as with NO. However, the mediators involved in inflammation of nerve due to mechanical compression remain almost unknown. METHODS: In this study, the median nerve of dogs was compressed with a clip for three weeks to observe the changes caused by compression. Immunohistochemistry was done by the avidin-biotin-peroxidase complex method to observe the changes of T cells (CD45) and macrophages (Mac-1) after compression. Antibodies against IL-1beta, TNF-alpha, and inducible nitric oxide synthesis (i-NOS) were used to examine the localization and changes of these mediators caused by nerve compression. RESULTS: In control animals, resident T cells were detected, but there were no macrophages. IL-1beta was positive in the Schwann cells and vascular endothelial cells. However, no cells showed TNF-alpha or i-NOS positively. After nerve compression, numerous T cells and macrophages appeared among the demyelinized nerve fibers. The macrophages were positive for IL-1beta, TNF-alpha and i-NOS. CONCLUSION: Inflammatory cytokines and NO may be involved in intraneural inflammatory changes arising from mechanical compression. Such mediators may be of importance in the manifestation of neuropathy.