Toe Spread Reflex Research Articles

Skeletal muscle reprogramming enhances reinnervation after peripheral nerve injury

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. PNI is characterized by nerve degeneration distal to the site of nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of “accepting” innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generate a mouse model in which NANOG, a pluripotency-associated transcription factor is expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulates the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression leads to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation. Further, NANOG mice demonstrate extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice show greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI.

Contribution of CD137-CD137L pathway in sciatic nerve crush-induced behavioral hypersensitivity and functional reduction

Abstract The T cell costimulatory molecule CD137L is involved in the development of neuropathic pain following peripheral nerve injury. Previously, our work has shown that absence or neutralization of CD137L reduces hypersensitivity, sensory and functional impairment following a sciatic nerve crush (SNC). CD137L activation utilizes both a CD137-dependent and a CD137-independent pathway. To further determine the contribution of these pathways, we examined pain-like and functional recovery-related behavioral responses following intrathecal injection (IT) of a CD137 neutralizing antibody (αCD137; daily days 3–10) after SNC in male and female B6 mice. Mice were randomly assigned to 1 of 4 groups: PBS, 0.5μg isotype control, 0.1 or 0.5μg of αCD137, and then were evaluated for sensitivity (von Frey, Hargreaves, & cold plantar test), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functions before surgery and on post-surgery days 1–77. On post-SNC day 1, all groups showed changes in sensitivity and decreased sensory and motor functions. αCD137 treatment did not alter mechanical, heat or hypersensitivity compared to PBS- or isotype-treated controls. αCD137-treated groups showed faster sensory (pin prick score) and motor functional recovery (toe spread reflex, toe spacing), as well as slightly greater improvement in grip strength test compared to controls. Together, our results indicate partial involvement of the CD137-dependent pathway in SNC-induced sensory and motor function changes following peripheral nerve injury. (Supported by NIH/NINDS R01NS098426 (Cao))

Early treatment of a CD137L neutralizing antibody decreases mechanical hypersensitivity and functional recovery time following a sciatic nerve crush

Abstract To investigate the role of CD137L in pain development and nerve regeneration following peripheral nerve injury, we examined pain-like and functional recovery-related behavioral responses following intrathecal injection (IT) of a CD137L neutralizing antibody (αCD137L; daily day 0–7) after sciatic nerve crush (SNC) in male and female B6 mice. Mice were randomly assigned to 1 of 4 groups: no IT, PBS, 0.1 or 0.5μg of αCD137L, and then were evaluated for sensitivity (von Frey, Hargreaves, & cold plantar test), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional recovery before surgery and on post-surgery days 1–77. On post-SNC day 1, all groups showed changes in sensitivity and decreased sensory and functional responses. By day 7, αCD137L treated mice showed reduced mechanical hypersensitivity compared to no IT and PBS-treated controls and were at baseline levels by day 42, about 1–2 weeks earlier than both control groups. All groups showed hyposensitivity to heat stimulus on day 1, but quickly returned to baseline with no group differences. On days 1–35 hyposensitivity to a cold stimulus was also found in all groups, but αCD137Ltreated mice were slightly more sensitive on days 49–77. Grip strength improved in all groups on day 14 without significant group differences. On days 3–14, αCD137L-treated groups had higher toe space scores compared to controls. Finally, sensory recovery (pin prick) in αCD137L-treated groups emerged on day 3, a week earlier than the control groups. Together, our results suggest that CD137L likely contributes to sensory hypersensitivity and functional changes early on following peripheral nervy injury.

(227) HIV Sensory Neuropathy Related Behaviors in Doxycycline Inducible HIV-1 Tat Transgenic Mice

Peripheral neuropathies are the most frequent neurological complications associated with HIV, within which HIV sensory neuropathy (SN) is the most common form, which affects 30-50% of HIV/acquired immunodeficiency syndrome (AIDS) patients. HIV SN is frequently manifested with hard-to-manage pain and is often under-diagnosed and/or under-treated. Tat, trans-activator of transcription, is a key activator of HIV transcription. Despite that recent studies have highlighted the role of HIV Tat in the pathophysiology of HIV-associated neurocognitive disorders (HANDs), the involvement of HIV Tat in neuropathic pain associated with HIV SN has not been extensively investigated. Our preliminary data showed that both single and repeated intrathecal injection(s) of HIV Tat induced mechanical hypersensitivity. In the current study, we further examined pain and functional related behavioral changes in doxycycline inducible HIV-1 Tat transgenic (iTat Tg) mice following the induction of Tat expression. Two induction regimen were compared: 7-day daily injection vs. 21-day daily injection of doxycycline (i.p. at 100mg/kg). Sensory sensitivities (mechanical, heat and cold) were assessed to evaluate pain-like behaviors. Sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional tests were used to evaluate hind paw functions associated with Tat expression. Both regimens induced significant mechanical hypersensitivity and significant reduction of grip strength from day 3 post the initiation of Tat induction to the end of the experiment (day 35) in iTat Tg mice. Tat induction for 21 days also significantly induced cold hypersensitivity in iTat Tg mice up to day 14 post-the first doxycycline injection. Neither regimen induced significant heat hypersensitivity or affected other functional measurements. Together, our results suggest the utility of iTat Tg mice in studying the role of Tat in HIV SN. Sex-depended differences and Tat-induced molecular pathways will be further investigated in future studies. (Grant support: R21 DA044886 [PI Cao])

Absence of CD137L decreases mechanical hypersensitivity and functional recovery time following sciatic nerve crush

Abstract CD137 Ligand (CD137L), a tumor necrosis factor superfamily molecule, is involved in immune responses such as migration, proliferation and production of cytokines. To investigate the involvement of CD137L in pain development and nerve regeneration following peripheral nerve injury, we examined various pain-like and functional recovery-related behavioral responses in CD137L knockout (KO) mice and wild type (WT) B6 mice following sciatic nerve crush (SNC). Male and female B6 WT and CD137LKO mice underwent a sham or SNC surgery and were evaluated for hypersensitivity (von Frey, Hargreaves & cold water tail withdrawal tests), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional recovery. Behaviors were assessed before surgery and on post-surgery days 1–77. On post-SNC Day 1, both KO and WT mice showed increased hypersensitivity and decreased sensory and functional responses. By Day 7, CD137LKO mice showed reduced mechanical hypersensitivity compared to WT and were responding at sham levels by Day 42, whereas WT controls reached sham levels by Days 56–63. Lack of CD137L did not alter warm or cold thermal hypersensitivity. Grip strength improved in both strains beginning on Day 7, but CD137LKO mice reached sham levels faster than WT controls (Day 17 vs. Day 70). No differences were found on toe spread reflex and toe spacing scores. Finally, pin prick scores revealed that sensory recovery in both strains of mice emerged on Day 7, but the CD137LKO mice responded similar to shams by Day 28, one week earlier than WT. Together, these results suggest that CD137L mediates the sensory hypersensitivity and impedes the restoration of sensory and motor functions following peripheral nerve injury.

Perturbation to Cholesterol at the Neuromuscular Junction Confers Botulinum Neurotoxin A Sensitivity to Neonatal Mice.

Botulinum neurotoxin A (BoNT/A) cleaves SNAP25 at the motor nerve terminals and inhibits stimulus evoked acetylcholine release. This causes skeletal muscle paralysis. However, younger neonatal mice (<P7; <7-days old) are resistant to the neuroparalytic effects of BoNT/A. That is, invivo injection of BoNT/A at the innervations of Extensor digitorum longus muscle in the hindlimbs inhibited the toe spread reflex within 24 hours following BoNT/A injection in adult mouse and in older (>P7) mice. However, neonatal mice younger than 7 days-age remained unaffected by BoNT/A injection. Also, BoNT/A inhibited stimulus evoked acetylcholine release and stimulus-evoked twitch tension of diaphragm nerve muscle preparations (NMPs) of adult mouse and >P7 neonates but not that of <P7. Moreover, NMPs of <P7 showed decreased uptake of fluorescent BoNT/A compared to >P7. However, cholesterol depletion using methyl-β-cyclodextrin (MβCD) sensitized <P7 neonates to BoNT/A and facilitated BoNT/A uptake into NMPs obtained from <P7 neonates. Further, MβCD (10 mM; 30 min pretreatment) increased the interaction between synaptic vesicle protein 2 and BoNT/A. Also, cholesterol depletion increased the miniature endplate current in adult NMPs. Interestingly, cholesterol replenishment, invitro, delayed the onset of inhibitory effect of BoNT/A. Collectively, our data suggest that cholesterol rich lipid microdomains are involved in BoNT/A uptake mechanisms during development. Our data demonstrate that cholesterol depletion sensitized neonatal mice (<P7) to BoNT/A while replenishing cholesterol delayed the onset of inhibitory actin of BoNT/A. This suggests that membrane cholesterol modulates neurotoxin sensitivity at the neuromuscular junction (NMJ).

In vitro and in vivo optimization of infrared laser treatment for injured peripheral nerves

Repair of peripheral nerve injuries remains a major challenge in restorative medicine. Effective therapies that can be used in conjunction with surgical nerve repair to improve nerve regeneration and functional recovery are being actively investigated. It has been demonstrated by a number of peer reviewed publications that photobiomodulation (PBM) supports nerve regeneration, reinnervation of the denervated muscle, and functional recovery after peripheral nerve injury. However, a key issue in the use of PBM as a treatment for peripheral nerve injury is the lack of parameter optimization for any given wavelength. The objective of this study was to demonstrate that for a selected wavelength effective in vitro dosing parameters could be translated to effective in vivo parameters. Comparison of infra-red (810 and 980 nm wavelengths) laser treatment parameters for injured peripheral nerves was done beginning with a series of in vitro experiments using primary human fibroblasts and primary rat cortical neurons. The primary rat cortical neurons were used for further optimization of energy density for 980 nm wavelength light using measurement of total neurite length as the bioassay. For these experiments, the parameters included a 1 W output power, power density of 10 mW/cm(2) , and energy densities of 0.01, 0.1, 0.5, 2, 10, 50, 200, 1,000, and 5,000 mJ/cm(2) . For translation of the in vitro data for use in vivo it was necessary to determine the transcutaneous penetration of 980 nm wavelength light to the level of the peroneal nerve. Two anesthetized, male White New Zealand rabbits were used for these experiments. The output power of the laser was set at 1.0 or 4.0 W. Power density measurements were taken at the surface of the skin, sub-dermally, and at the level of the nerve. Laser parameters used in the in vivo studies were calculated based on data from the in vitro studies and the light penetration measurements. For the in vivo experiments, a total of 22 White New Zealand rabbits (2.34-2.89 kg) were used. Translated dosing parameters were refined in a pilot study using a transection model of the peroneal nerve in rabbits. Output powers of 2 and 4 W were tested. For the final set of in vivo experiments, the same transection nerve injury model was used. An energy density of 10 mW/cm(2) at the level of the peroneal nerve was selected and the laser parameters were further refined. The dosing parameters used were: 1.5 W output power, 43 seconds exposure, 8 cm(2) area and a total energy of 65 J. In vitro, 980 nm wavelength light at 10 mW/cm(2) significantly improved neurite elongation at energy densities between 2 and 200 mJ/cm(2) . In vivo penetration of the infrared light measured in anesthetized rabbits showed that on average, 2.45% of the light applied to the skin reached the depth of the peroneal nerve. The in vivo pilot study data revealed that the 4 W parameters inhibited nerve regeneration while the 2 W parameters significantly improved axonal regrowth. For the final set of experiments, the irradiated group performed significantly better in the toe spread reflex test compared to the control group from week 7 post-injury, and the average length of motor endplates returned to uninjured levels. The results of this study demonstrate that treatment parameters can be determined initially using in vitro models and then translated to in vivo research and clinical practice. Furthermore, this study establishes that infrared light with optimized parameters promotes accelerated nerve regeneration and improved functional recovery in a surgically repaired peripheral nerve.

Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions

Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875 – fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.

Capsaicin interacts with clathrin coated pit dependent mechanisms to protect motor nerve terminals against botulinum neurotoxin A

Botulinum neurotoxin A (BoNT/A) cleaves SNAP‐25 and terminates acetylcholine release at the motor nerve endings (MNEs) resulting in neuroparalysis. Recently, we reported that capsaicin, a TRPV1 channel agonist, protected MNEs from the inhibitory actions of BoNT/A (Thyagarajan et al 2009). This study examines the mechanisms of capsaicin's prophylactic action against BoNT/A. In control mice injection of BoNT/A (3 μl; 6.67 pM) near the innervations of the Extensor digitorum longus muscle of both hind limbs inhibited the toe spread reflex (TSR) within 24 hrs. However, when capsaicin (3 μl; 1 mM) was injected either 5 to10 minutes or 4 or 8 hours before BoNT/A, the TSR remained normal. Capsaicin pretreatment in vitro significantly reduced fluorescent BoNT/A uptake into MNEs and protected stimulus evoked twitch tension and acetylcholine release from MNEs while BoNT/A alone inhibited these motor nerve functions. Capsazepine, a TRPV1 antagonist prevented the protective effects of capsaicin. To understand the mechanism of capsaicin‐induced protection against BoNT/A, we studied cholinergic Neuro 2a cells. Acute exposure to capsaicin altered the subcellular localization of clathrin heavy chain (Chc) and AP2, two proteins essential to clathrin coated pit (CCP) formation. Chlorpromazine, an inhibitor of CCP dependent endocytosis (Maksymowych and Simpson, 2004), mimicked the effects of capsaicin on Chc delocalization. Wortmannin, (WMN, nonselective PI5K inhibitor), reduced fluorescent BoNT/A uptake into MNEs and delocalized Chc and AP2 in Neuro 2a cells. WMN (3 μl; 1 mM) injected 6 hours before BoNT/A protected mice from neuroparalysis. We hypothesize that capsaicin reduced the neuroparalytic effects of BoNT/A by inhibiting CCP dependent uptake of BoNT/A into MNEs.

Effects of hydroxamate metalloendoprotease inhibitors on botulinum neurotoxin A poisoned mouse neuromuscular junctions.

Currently the only therapy for botulinum neurotoxin A (BoNT/A) poisoning is antitoxin. Antidotes that are effective after BoNT/A has entered the motor nerve terminals would dramatically benefit BoNT/A therapy. Inhibition of proteolytic activity of BoNT/A light chain by metalloendoprotease inhibitors (MEIs) is under development. We tested the effects of MEIs on in vitro as well as in vivo BoNT/A poisoned mouse nerve-muscle preparations (NMPs). The K(i) for inhibition of BoNT/A metalloendoprotease was 0.40 and 0.36 muM, respectively, for 2,4-dichlorocinnamic acid hydroxamate (DCH) and its methyl derivative, ABS 130. Acute treatment of nerve-muscle preparations with 10 pM BoNT/A inhibited nerve-evoked muscle twitches, reduced mean quantal content, and induced failures of endplate currents (EPCs). Bath application of 10 muM DCH or 5 muM ABS 130 reduced failures, increased the quantal content of EPCs, and partially restored muscle twitches after a delay of 40-90 min. The restorative effects of DCH and ABS 130, as well as 3,4 diaminopyridine (DAP) on twitch tension were greater at 22 degrees C compared to 37 degrees C. Unlike DAP, neither DCH nor ABS 130 increased Ca(2+) levels in cholinergic Neuro 2a cells. Injection of MEIs into mouse hind limbs before or after BoNT/A injection neither prevented the toe spread reflex inhibition nor improved muscle functions. We suggest that hydroxamate MEIs partially restore neurotransmission of acutely BoNT/A poisoned nerve-muscle preparations in vitro in a temperature dependent manner without increasing the Ca(2+) levels within motor nerve endings.

Capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of botulinum neurotoxin a.

Botulinum neurotoxin A (BoNT/A), the most toxic, naturally occurring protein, cleaves synapse-associated protein of 25 kDa and inhibits acetylcholine release from motor nerve endings (MNEs). This leads to paralysis of skeletal muscles. Our study demonstrates that capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of BoNT/A. Bilateral injection of BoNT/A near the innervation of the Extensor digitorum longus (EDL) muscle of adult Swiss-Webster mice inhibited the toe spread reflex (TSR). However, when capsaicin was coinjected bilaterally, or injected 4 or 8 h before injecting BoNT/A, the TSR remained normal. In animals that were pretreated with capsazepine, capsaicin failed to protect against the neuroparalytic effects of BoNT/A. In vivo analyses demonstrated that capsaicin protected muscle functions and electromygraphic activity from the incapacitating effects of BoNT/A. The twitch response to nerve stimulation was greater for EDL preparations isolated from mice injected with capsaicin before BoNT/A. Capsaicin pretreatment also prevented the inhibitory effects of BoNT/A on end-plate currents. Furthermore, pretreatment of Neuro 2a cells with capsaicin significantly preserved labeling of synaptic vesicles by FM 1-43. This protective effect of capsaicin was observed only in the presence of extracellular Ca(2+) and was inhibited by capsazepine. Immunohistochemistry demonstrated that MNEs express transient receptor potential protein of the vanilloid subfamily, TRPV1, the capsaicin receptor. Capsaicin pretreatment, in vitro, reduced nerve stimulation or KCl-induced uptake of BoNT/A into motor nerve endings and cholinergic Neuro 2a cells. These data demonstrate that capsaicin interacts with TRPV1 receptors on MNEs to reduce BoNT/A uptake via a Ca(2+)-dependent mechanism.

Capsaicin Protects Mouse Neuromuscular Junctions From The Paralytic Effects Of Botulinum Neurotoxin A

Botulinum neurotoxins (BoNTs) are the most toxic naturally occurring proteins. Botulinum serotype A (BoNT/A) selectively cleaves SNAP-25 and inhibits acetylcholine release from motor nerve endings resulting in life threatening paralysis of skeletal muscles. Here we report that capsaicin (8-methyl-N-vanillyl-6-nonenamide), an irritant active principle of chili peppers, protects against the paralytic effects of BoNT/A in mouse. In Triangularis sterini nerve muscle preparations, capsaicin pretreatment in vitro, significantly reduced fluorescent labeled BoNT/A uptake mediated by depolarization with 40 mM KCl or neural stimulations. In vivo injection of capsaicin (3 μl of a 1 mM stock solution) either coinjected or injected 4 or 8 hours before BoNT/A injection protected the mice from the inhibitory effects of BoNT/A measured by toe spread reflex. In controls the toe spreading reflex was inhibited within 24 hrs of poisoning with BoNT/A. Also wortmannin, a PIP5K inhibitor, injected prior to BoNT/A exposure, protected the mice from the paralytic effect of BoNT/A. In vitro muscle tension measurements demonstrate that capsaicin pretreatment partially protected the functions of the mouse Extensor digitorum longus nerve muscle preparations. Also pretreatment of cultured cholinergic neuroblastoma (N2a) cells with 10 μM capsaicin for 10 minutes prior to exposure of 10 pM BoNT/A significantly preserved labeling of synaptic vesicle pools by FM1-43. Our data collectively demonstrate that capsaicin offers protection from the paralyzing effect of BoNT/A by significantly reducing the uptake of the toxin in mouse neuromuscular junction probably by interfering with endocytosis of the toxin.Supported by HDTRA1-07-C-0031 CBT BAA.

Anatomical and Functional Outcomes following a Precise, Graded, Dorsal Laceration Spinal Cord Injury in C57BL/6 Mice

To study the pathophysiology of spinal cord injury (SCI), we used the LISA-Vibraknife to generate a precise and reproducible dorsal laceration SCI in the mouse. The surgical procedure involved a T9 laminectomy, dural resection, and a spinal cord laceration to a precisely controlled depth. Four dorsal hemisection injuries with lesion depths of 0.5, 0.8, 1.1, and 1.4 mm, as well as normal, sham (laminectomy and dural removal only), and transection controls were examined. Assessments including the Basso Mouse Scale (BMS), footprint analysis, beam walk, toe spread reflex, Hargreaves' test, and transcranial magnetic motor-evoked potential (tcMMEP) analysis were performed to assess motor, sensorimotor, and sensory function. These outcome measures demonstrated significant increases in functional deficits as the depth of the lesion increased, and significant behavioral recovery was observed in the groups over time. Quantitative histological examination showed significant differences between the injury groups and insignificant lesion depth variance within each of the groups. Statistically significant differences were additionally found in the amount of ventral spared tissue at the lesion site between the injury groups. This novel, graded, reproducible laceration SCI model can be used in future studies to look more closely at underlying mechanisms that lead to functional deficits following SCI, as well as to determine the efficacy of therapeutic intervention strategies in the injury and recovery processes following SCI.

Capsaicin protects neuromuscular junctions from the inhibitory effects of botulinum neurotoxin A

AbstractWithin 24 hrs after injecting botulinum neurotoxin A (BoNT/A) into the hindlimb, mice lost the toe spread reflex and developed progressive muscle weakness. At the same time, the compound muscle action potential amplitude decreased. Injection of capsaicin before BoNT/A significantly reduced these affects and protected the muscle twitch tension of the Extensor digitorum longus (EDL) nerve muscle preparation. Acute in vitro exposure of isolated nerve muscle preparations, as well as Neuro 2a cells, to capsaicin prevented uptake of Alexa 647 BoNT/A. Motor nerve endings as well as Neuro 2a cells express the capsaicin receptor, a transient receptor potential channel of the vanilloid family (TRPV1). Capsaicin as well as disruption of clathrin coated pits (CCPs) reduced Neuro 2a cell uptake of BoNT/A. FM1-43 uptake indicated that exocytosis persists for BoNT/A treated Neuro 2a cells pretreated with capsaicin. Pre-injection of wortmannin (WMN), a PI3Kinase inhibitor, also protected mice from the paralytic effects of BoNT/A. When applied alone, either WMN or capsaicin selectively reduced stimulus-evoked transmitter release from motor nerve endings. We hypothesize that TRPV1 activation reduces PI(4,5)P2 level within the membrane. This prevents CCP formation and uptake of BoNT/A.

Intermittent reperfusion fails to prevent posttourniquet neurapraxia.

This study examined the effects of intermittent reperfusion on peripheral nerve function. Rabbits were randomized to undergo 4 hours of 350 mm Hg tourniquet compression to a hind limb either continuously, interrupted by a single 10-minute reperfusion interval after 2 hours, or interrupted by 10 minutes of reperfusion after each hour. A control group had the tourniquet applied for 4 hours but it was never inflated. The animals were examined clinically for neuromuscular dysfunction and the structure and function of the peripheral nerves were evaluated 1 week after tourniquet compression. Animals that underwent compression had a foot drop and decreased toe-spread reflex. There was greater intraneural edema and slower nerve conduction velocity in nerve segments that were directly compressed by the tourniquet but no apparent abnormalities in segments distal to the tourniquet. Intermittent reperfusion failed to diminish the clinical, structural, or functional consequence of the neurologic injury.

Peripheral nerve elongation by laser Doppler flowmetry controlled expansion: Functional and neurophysiological aspects

A new method for elongation of peripheral nerves with preservation of function is presented. Nerve blood flow during experimental nerve expansion of rabbit sciatic nerve is controlled by laser Doppler flowmetry in order to avoid nerve ischemia. Using this method, nerve function in relation to gait remained intact in 72.5% of the animals and recovered within 3 weeks in the others. Disturbances in toe-spread reflex recovered completely in all animals after 3 weeks. Significant nerve elongation up to 40% is possible with preservation of function when nerve blood flow is controlled by laser Doppler flowmetry.

Nerve function and structure beneath and distal to a pneumatic tourniquet applied to rabbit hindlimbs.

Neurophysiologic and neuropathologic changes were studied in rabbit hindlimbs after 2 hours of pneumatic tourniquet application with either 350 mmHg (n = 18) or 1,000 mmHg (n = 6) cuff inflation pressure. The toe spread reflex was decreased in 66% and absent in 33% of limbs 2 days after 350 mmHg compression, and was absent in all limbs after 1,000 mmHg compression. Compound motor action potential amplitudes (CMAPs), recorded from the abductor hallucis muscle, were significantly decreased with sciatic nerve stimulation 1 hour after 350 mmHg compression. CMAPs returned to baseline values one and two days later, however nerve conduction velocity (NCV) was still significantly decreased in the compressed sciatic nerves of these groups. In contrast, complete nerve conduction block, localized beneath the cuff's distal border, was observed two days after 1,000 mmHg compression, and NCV was still significantly decreased distal to the tourniquet zone. Using light and electron microscopy, scattered axonal degeneration, mild myelin damage, and normal nodes of Ranvier were observed two days after 350 mmHg tourniquet compression. Severe fiber damage and nodal obliteration were noted after 1,000 mmHg tourniquet compression. Although nodal invagination is probably not a significant pathogenic mechanism at clinically relevant tourniquet pressures and durations, functional abnormalities were induced by 2 hour, 350 mmHg tourniquet compression. Such changes probably correlate with clinical electromyographic abnormalities and delayed post-operative recovery following 'routine' extremity surgery using pneumatic tourniquets.

Problems with the use of the toe-spreading reflex in rats as an assay in nerve regeneration studies

Stages in the return of the toe-spreading reflex after sciatic nerve injury were examined using the rat. It was found that the earliest stages in the return of the reflex do not indicate nerve regeneration, but rather reflect the development of adrenalin sensitivity in denervated muscles. Probably systemic adrenalin released during the reflex-testing procedure causes muscle contractions which imitate a nerve-induced toe-spread reflex.