建立人工面神经反射弧恢复失神经支配眼轮匝肌的闭眼功能

目的：建立人工面神经反射弧,恢复面瘫兔的闭眼功能。 方法：试验动物为新西兰兔12只。采用手术切断面神经制备单侧周围性面瘫模型,患侧行肌电图检查确诊。术后第5天患侧植入刺激电极,健侧植入采集电极并留置1月。患侧分别于术后第7天、第28天给予电流刺激;而健侧采集电极则连续采集2周的肌电信号。建立健侧眼轮匝肌肌电信号采集、中枢信号处理模式识别、患侧电流刺激眼轮匝肌系统。当健侧眼轮匝肌采集的肌电信号经信号识别、提取以及电脑分析判断,符合其闭眼刺激阈值时,即对人工电刺激器发出指令,由刺激电极直接作用于患侧眼轮匝肌,引起眼睑完全闭合。结果：刺激方式为正负方向矩形波,电流强度为0.40—0.70mA之间可引起患侧眼轮匝肌收缩,眼睑完全闭合。当健侧眼睑闭合时,其肌电信号电压大于50uV,触发电刺激器,电流刺激即引发患侧眼轮匝肌收缩,完成眼睑闭合。结论：利用MEMS技术,在面瘫兔模型建立“人工面神经反射弧”,恢复患侧眼轮匝肌闭眼功能。     

Motor unit number index (MUNIX) in the orbicularis oculi muscle of healthy subjects

Introduction: The motor unit number index (MUNIX) refers to an electrophysiological method that measures the number of motor units in the surface electromyographic interference pattern (SIP) recorded during graded muscle contractions. MUNIX studies of limb muscles have been conducted, but MUNIX studies of bulbo‐facial muscles have not been reported. Methods: We assessed bilateral orbicularis oculi muscles using MUNIX, and the reference values and reproducibility of MUNIX and motor unit size index (MUSIX) were investigated in healthy subjects. Results: In this study, MUNIX was applied successfully to the orbicularis oculi muscles and showed good reproducibility. The correlation coefficients for MUNIX and MUSIX were 0.803 and 0.592, respectively, and the coefficients of variation were 20.9% and 8.5%, respectively. Conclusions: The MUNIX procedure for the orbicularis oculi muscle would be a useful tool for evaluating bulbar symptoms, especially in amyotrophic lateral sclerosis. Muscle Nerve 51 : 197–200, 2015     

Serial SFEMG studies of orbicularis oculi muscle after the first administration of botulinum toxin

Serial single fiber electromyography (SFEMG) examinations of orbicularis oculi muscle in patients with blepharospasm or hemifacial spasm treated with botulinum toxin injections were performed. The aim of the study was to evaluate the impairment of neuromuscular transmission, to follow reinnervation after botulinum toxin administration and to find out whether there was a relationship between SFEMG parameters and clinical symptoms. Examinations were performed before injection, during early and late remission of symptoms, and after recurrence of the involuntary movement. Severe impairment of neuromuscular transmission, as revealed by increased jitter and increased presence of abnormal potential pairs and pairs with blocking, was found in early remission, but fiber density remained unchanged when compared with pretreatment values. In late remission, increased fiber density was registered for the first time. The recurrence of involuntary movements was related to the further increase of fiber density and tendency to normalization of jitter parameters. The study therefore suggests that formation of new neuromuscular junctions and their functional maturation is responsible for muscle recovery after botulinum toxin administration.     

Ocular myastyenia gravis: The diagnostic yield of repetitive nerve stimulation and stimulated single fiber EMG of orbicularis oculi muscle and infrared reflection oculography

For the diagnosis of ocular myasthenia gravis (ocular MG), testing of the muscles close to the affected ones may be important. The relative importance of several methods: stimulated single fiber EMG (stimulated SFEMG), repetitive nerve stimulation test (RNS) of orbicularis oculi muscle, and infrared reflection oculography (IROG) was investigated. Thirty-two patients in whom a diagnosis of ocular MG was considered on clinical grounds were admitted to the study. Based on the results of the three neurophysiological tests, the patients could be divided in three groups: a first group with an abnormal stimulated SFEMG, and an abnormal RNS and/or abnormal IROG; a second group with only a slightly abnormal stimulated SFEMG; and a third group with normal tests in all three tests. The clinical diagnosis of ocular MG was made in all 11 patients of the first group; in 86% (6 of 7) of the patients of the second group; and in 7% (1 of 14) of the patients of the third groups. This study demonstrates that the orbicularis oculi muscle is a suitable muscle for stimulated SFEMG in patients with ocular MG, and that the results obtained with this technique showed a better relation with the clinical diagnosis than those of the two other techniques. We also demonstrate that there is no additional value in studying the jitter with different stimulation rates in patients with suspected ocular MG. © 1993 John Wiley & Sons, Inc.     

眼轮匝肌肌电信号在不同运动模式下的定量特征信息

Objective: To research function of rbicularis oculi muscle (OOM) and explore the relationship between the electromyography (EMG) signals and the functional actions of the muscle via analyzing the EMG signals features of OOM of rabbit in different movement states. Methods: The EMG signals were extracted from OOM of normal rabbits by the implanted microelectrodes. Then the features of these EMG signals were analyzed in the time domain and the frequency domain. Results: The average peak values of the absolute EMG amplitude for natural continuous eye-opening event, natural continuous eye-closing state, natural eye-blinking movement and evoked eye-closing state were 29.3μV, 34.7μV, 435.2μV and 737.3μV respectively. The peak frequency values of the EMG power spectrum for the four modes of OOM were 99Hz, 143Hz, 193Hz and 374Hz respectively. The electrical activities of OOM during the natural eye-opening event and the natural continuous eye-closing state were stable and displayed low amplitudes. During the spontaneous blink state and the evoked eye-closing state, the EMG amplitudes markedly increased, and the increased level in the latter state was stronger than that in the former state. When rabbits continuously closed eye or opened eye, all of the peak values of the absolute voltage amplitude were less than 50μV.The absolute amplitude values of the starting site were between 50μV and 60μV during the spontaneous blink and the evoked eye-closing movements. The whole frequency band of the energy of the power spectrum density (PSD) of OOM was between 0Hz and 500Hz, and the primary frequency range was between 20Hz and 350Hz.In the time domain, the difference was not significant for the EMG signals of OOM between the continuous eye-opening state and the continuous eye-closing movement, but there were statistically significant in the other states for their pairwise comparisons. In the frequency domain, there was no significant difference for the peak frequency of PSD about the EMG signals when compared the continuous eye-opening state with the continuous eye-closing event or compared the continuous eye-closing state with the spontaneous blink movement. When compared that in the other movements with each other, however, the differences were significant. Conclusions: OOM relaxes when eyelid keeps continuously opening. The action of eyelid-closing is due to contraction of this muscle. Each state has its own features of the EMG signals for OOM, these features can be as foundation for computer to judge and identify movement states of the muscle.     

Changes in a rat facial muscle after facial nerve injury and repair

This study describes changes in a rat facial muscle innervated by the mandibular and buccal facial nerve branches 4 months after nerve injury and repair. The following groups were studied: (A) normal controls; (B) spontaneous reinnervation by collateral or terminal sprouting; (C) reinnervation after surgical repair of the mandibular branch; and (D) chronic denervation. The normal muscle contained 1200 exclusively fast fibers, mainly myosin heavy chain (MyHC) IIB fibers. In group B, fiber number and fiber type proportions were normal. In group C, fiber number was subnormal. Diameters and proportions of MyHC IIA and hybrid fibers were above normal. The proportion of MyHC IIB fibers was subnormal. Immediate and delayed repair gave similar results with respect to the parameters examined. Group D rats underwent severe atrophic and degenerative changes. Hybrid fibers prevailed. These data suggest that spontaneous regeneration of the rat facial nerve is superior to regeneration after surgical repair and that immediacy does not give better results than moderate delay with respect to surgical repair. Long delays are shown to be detrimental.     

Acute morphologic changes in orbicularis oculi muscle after doxorubicin injection into the eyelid

This study attempts to gain a better understanding of the primary cause of doxorubicin myotoxicity to aid in the development of a protocol to increase its effectiveness in treating muscle spasm diseases. The time course of acute injury to the orbicularis oculi muscle after injection of doxorubicin into the eyelids of rabbits was examined. The effects of doxorubicin injection were immediate and dramatic. Within 5 minutes, the muscle cells in the eyelids showed signs of myofibrillar dissolution. This process continued for the first 24 hours, with a marked decrease in the total number of myofibers by 1 hour after drug treatment. By 4 days after doxorubicin injection, most of the treated muscle had a more normal morphology, although some ongoing myofibrillar dissolution was present. There was evidence for a limited amount of muscle regeneration. Three levels of injury were seen: a severe, irreversible injury in the majority of the myofibers; a mild, reversible injury in a subset of the fibers; and a group of myofibers apparently uninjured as a result of doxorubicin treatment. Doxorubicin toxicity in skeletal muscle is of rapid onset. Understanding the primary cause and acute changes in doxorubicin myotoxicity may accelerate the development of improved protocols to increase the effectiveness of doxorubicin in treating muscle spasm diseases.     

Morphological changes of Golgi apparatus in adult rats after facial nerve injuries

We examined the morphological changes of Golgi apparatus (GA) of the facial motor neurons in rats after facial nerve avulsion or axotomy. In rats after avulsion, the numbers of motor neurons showed reduction and fragmentation of GA, namely the organelle lost the normal network‐like configuration which was replaced by numerous small disconnected elements (fine fragmentation). This GA fragmentation was morphologically indistinguishable from that previously reported in amyotrophic lateral sclerosis (ALS). On the other hand, axotomy did not induce significant motor neuron loss, and the GA had lost the elongated profiles (coarse fragmentation). These results suggest that there may be a similar cascade leading to motor neuron death in rats after avulsion, and ALS and GA observed in rats after axotomy may not be related to neuronal death.     

Accumulation of glycogen in axotomized adult rat facial motoneurons

This study biochemically determined glycogen content in the axotomized facial nucleus of adult rats up to 35 days postinsult. The amounts of glycogen in the transected facial nucleus were significantly increased at 5 days postinsult, peaked at 7 days postinsult, and declined to the control levels at 21–35 days postinsult. Immunohistochemical analysis with antiglycogen antibody revealed that the quantity of glycogen granules in the axotomized facial nucleus was greater than that in the control nucleus at 7 days postinjury. Dual staining methods with antiglycogen antibody and a motoneuron marker clarified that the glycogen was localized mainly in motoneurons. Immunoblotting and quantification analysis revealed that the ratio of inactive glycogen synthase (GS) to total GS was significantly decreased in the injured nucleus at about 1–3 days postinsult and significantly increased from 7 to 14 days postinsult, suggesting that glycogen is actively synthesized in the early period postinjury but suppressed after 7 days postinsult. The enhanced glycogen at about 5–7 days postinsult is suggested to be responsible for the decrease in inactive GS levels, and the decrease of glycogen after 7 days postinsult is considered to be caused by increased inactive GS levels and possibly the increase in active glycogen phosphorylase. © 2015 Wiley Periodicals, Inc.     

Stimulated single-fiber EMG of the frontalis and orbicularis oculi muscles in ocular myasthenia gravis

We performed single-fiber electromyography by axonal stimulation (stimulated SFEMG) of the frontalis and orbicularis oculi muscles of 20 patients with ocular myasthenia gravis (OM) and 46 controls. In controls, mean consecutive differences (MCD) ranged from 5 to 55 micros (average, 14.7 +/- 2.8 micros) in the frontalis and from 4 to 56 micros (average, 12.56 +/- 2.19 micros) in orbicularis oculi. The mean MCD of individual muscle potentials (MPs) was 14.6 +/- 6.8 micros in frontalis and 12.68 +/- 6.10 micros in orbicularis oculi. In the OM patients, the mean MCD was 43.85 +/- 25.18 micros in the frontalis and 69.85 +/- 29.55 micros in orbicularis oculi (P < 0.0001), and the number of MPs with altered MCD was 7.15 +/- 4.66 (range, 1-18) and 12.65 +/- 4.90 (range, 6-21), respectively (P < 0.0001). We conclude that stimulated SFEMG of the orbicularis oculi muscle is more sensitive for the diagnosis of OM than of the frontalis muscle.     

Afferent projections to the orbicularis oculi motoneuronal cell group. An autoradiographical tracing study in the cat

The motoneurons innervating the orbicularis oculi muscle from a subgroup within the facial nucleus, called the intermediate facial subnucleus. This makes it possible to study afferents to these motoneurons by means of autoradiographical tracing techniques. Many different injections were made in the brainstem and diencephalon and the afferent projections to the intermediate facial subnucleus were studied. The results indicated that these afferents were derived from the following brainstem areas: the dorsal red nucleus and the mesencephalic tegmentum dorsal to it; the olivary pretectal nucleus and/or the nucleus of the optic tract; the dorsolateral pontine tegmentum (parabrachial nuclei and nucleus of K?lliker-Fuse) and principal trigeminal nucleus; the ventrolateral pontine tegmentum at the level of the motor trigeminal nucleus; the caudal medullary medial tegmentum; the lateral tegmentum at the level of the rostral pole of the hypoglossal nucleus and the ventral part of the trigeminal nucleus and the nucleus raphe pallidus and caudal raphe magnus including the adjoining medullary tegmentum. These latter projections probably belong to a general motoneuronal control system. The mesencephalic projections are mainly contralateral, the caudal pontine and upper medullary lateral tegmental projections are mainly ipsilateral and the caudal medullary projections are bilateral. It is suggested that the different afferent pathways subserve different functions of the orbicularis oculi motoneurons. Interneurons in the dorsolateral pontine and lateral medullary tegmentum may serve as relay for cortical and limbic influences on the orbicularis oculi musculature, while interneurons in the ventrolateral pontine and caudal medullary tegmentum may take part in the neuronal organization of the blink reflex.     

A new method for measuring compound muscle action potentials in facial palsy: a preliminary study

To establish a simple, reproducible procedure for studying facial motor nerve conduction (MNC), we determined the optimal electrode position to record evoked compound muscle action potentials (CMAPs) from perioral muscles in normal subjects. We examined three new electrode positions in which the electrode connected to the one input of the amplifier was placed on the mental protuberance, and the one connected to the other input was placed on the skin over the orbicularis oris muscle (the philtrum, mouth angle, or lower lip). We then compared the morphology and amplitudes of the CMAPs, right-left differences, and the reproducibility of CMAP amplitudes with recordings taken from the standard electrode position in which one electrode was placed on the nasolabial fold closely lateral to the ala nasi, and the other was placed on the skin over the orbicularis oris. Percutaneous supramaximal electrical stimulation was applied to the main trunk of the facial nerve. All three of the new recording positions showed greater amplitudes and more obvious biphasic CMAPs than the standard method. Positioning the electrode connected to the negative input on the philtrum was optimal in terms of right-left differences and the reproducibility of CMAP amplitudes. Therefore, this midline recording is a simple, reproducible method for calculating the CMAP amplitude ratio. However, prior to clinical use of this procedure, analyses of patients with facial palsy are required.     

Differential gene expression in the axotomized facial motor nucleus of presymptomatic SOD1 mice

Previously, we compared molecular profiles of one population of wild-type (WT) mouse facial motoneurons (FMNs) surviving with FMNs undergoing significant cell death after axotomy. Regardless of their ultimate fate, injured FMNs respond with a vigorous pro-survival/regenerative molecular response. In contrast, the neuropil surrounding the two different injured FMN populations contained distinct molecular differences that support a causative role for glial and/or immune-derived molecules in directing contrasting responses of the same cell types to the same injury. In the current investigation, we utilized the facial nerve axotomy model and a presymptomatic amyotrophic lateral sclerosis (ALS) mouse (SOD1) model to experimentally mimic the axonal die-back process observed in ALS pathogenesis without the confounding variable of disease onset. Presymptomatic SOD1 mice had a significant decrease in FMN survival compared with WT, which suggests an increased susceptibility to axotomy. Laser microdissection was used to accurately collect uninjured and axotomized facial motor nuclei of WT and presymptomatic SOD1 mice for mRNA expression pattern analyses of pro-survival/pro-regeneration genes, neuropil-specific genes, and genes involved in or responsive to the interaction of FMNs and non-neuronal cells. Axotomized presymptomatic SOD1 FMNs displayed a dynamic pro-survival/regenerative response to axotomy, similar to WT, despite increased cell death. However, significant differences were revealed when the axotomy-induced gene expression response of presymptomatic SOD1 neuropil was compared with WT. We propose that the increased susceptibility of presymptomatic SOD1 FMNs to axotomy-induced cell death and, by extrapolation, disease progression, is not intrinsic to the motoneuron, but rather involves a dysregulated response by non-neuronal cells in the surrounding neuropil.     

Manual stimulation of facial muscles improves functional recovery after hypoglossal-facial anastomosis and interpositional nerve grafting of the facial nerve in adult rats

The facial nerve in humans is often prone to injuries requiring surgical intervention. In the best case, nerve reconstruction is achieved by a facial-facial anastomosis (FFA), i.e. suture of the proximal and distal stumps of the severed facial nerve. Although a method of choice, FFA rarely leads to a satisfactory functional recovery. We have recently devised and validated, in an established experimental paradigm in rats, a novel strategy to improve the outcome of FFA by daily manual stimulation (MS) of facial muscles. This treatment results in full recovery of facial movements (whisking) and is achieved by reducing the proportion of functionally detrimental poly-innervated motor end-plates. Here we asked whether MS could also be beneficial after two other commonly used surgical methods of clinical facial nerve reconstruction namely hypoglossal-facial anastomosis (HFA) and interpositional nerve grafting (IPNG) which, however, seem to have a poorer outcome compared to FFA. Compared to FFA, daily MS for 2 months after HFA and IPGN did not completely restore function but, nevertheless, significantly improved the amplitude of whisker movements by 50% compared with untreated animals. Functional improvement was associated with a reduction in the proportion of polyinnervated end-plates. MS did not reduce the extent of axonal branching at the lesion site nor the subsequent misdirected axonal regrowth to inappropriate targets. Our data show that a simple approach leading to improved quality of muscle fiber reinnervation is functionally beneficial after different types of clinically relevant surgical interventions.     

Neuroprotective effects of recombinant human erythropoietin on facial motoneurons after facial nerve injury in rats

BACKGROUND: Erythropoietin and recombinant human erythropoietin (rhEPO) inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can protect facial motoneurons (FMNs) as well.OBJECTIVE: To test the neuroprotective effects of rhEPO on injured FMNs, as well as the influence on Caspase-3 expression.DESIGN, TIME AND SETTING: Randomized, controlled, animal experiment. This study was performed at the Central Laboratory of Basic Medical College, Chongqing Medical University from January to October 2007.MATERIALS: Seventy-five female SD rats, weighing 210-230g. rhEPO injection was provided by Sansheng pharmaceuticals company, Shenyang City, Liaoning Province, China, and the License number was HMLN S20010001.METHODS: A total of 75 female rats were randomly divided into rhEPO treatment, control, and sham operation groups, with 25 rats in each group. Rat models of facial nerve injury were established in the rhEPO treatment group and the control group by crushing the main trunk of the left facial nerve. Surgical microscopic observation of the facial nerve damage displayed perineurial disruption. The left stylomastoid foramen of the sham operation group were only exposed, but without nerve injury. The rhEPO treatment group was treated with rhEPO (5000U/kg, i. p.) once following injury and once a day for two weeks. The control and sham operation groups were treated with the same dose of normal saline (i. p.), once following injury and once a day for two weeks.MAIN OUTCOME MEASURES: Rats were sacrificed 3, 7, 14, 21, and 28 days after injury, FMN survival after facial nerve injury was analyzed by Toluidine blue staining, and then survival ratios (L/R) were calculated. The number of apoptotic profiles in the injured FMNs were evaluated by TUNEL staining. Expression of Caspase-3 in the facial nucleus was detected by immunohistochemistry methods.RESULTS: A total of 75 rats were included in the final analysis. FMN survival ratios, both in rhEPO treatment group and control group, decreased gradually between seven and 28 days; however, FMN survival ratios were significantly greater in the rhEPO treatment group compared to the control group (P<0.05). No TUNEL-positive cells were observed three days after injury in the rhEPO treatment and control groups; however, by seven days after injury, apoptotic cells were observed and peaked by 14 days in the control group. Between seven and 21 days, apoptotic cell numbers were significantly lower in the rhEPO treatment group compared to the control group (P<0.05). The expression of Caspase-3 increased three days after injury and peaked at 14 days in the control group. Nevertheless, Caspase-3 expression was significantly lower in the rhEPO treatment group compared to the control group at each time point (P<0.05).CONCLUSION: Treatment with rhEPO can effectively protect facial motoneurons by reducing expression of Caspase-3 and inhibiting apoptosis.     

Neuroprotective effects of recombinant human erythropoietin on facial motoneurons after facial nerve injury in rats★

BACKGROUND： Erythropoietin and recombinant human erythropoietin （rhEPO） inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can protect facial motoneurons （FMNs） as Well. OBJECTIVE： To test the neuroprotective effects of rhEPO on injured VMNs, as well as the influence on Caspase-3 expression. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment. This study was performed at the Central Laboratory of Basic Medical College, Chongqing Medical University from January to October 2007. MATERIALS： Seventy-five female SD rats, weighing 210-230 g. rhEPO injection was provided by Sansheng pharmaceuticals company, Shenyang City, Liaoning Province, China, and the License number was HMLN S20010001. METHODS： A total of 75 female rats were randomly divided into rhEPO treatment, control, and sham operation groups, with 25 rats in each group. Rat models of facial nerve injury were established in the rhEPO treatment group and the control group by crushing the main trunk of the left facial nerve. Surgical microscopic observation of the facial nerve damage displayed perineurial disruption. The left stylomastoid foramen of the sham operation group were only exposed, but without nerve injury. The rhEPO treatment group was treated with rhEPO （5 000 U/kg, i.p.） once following injury and once a day for two weeks. The control and sham operation groups were treated with the same dose of normal saline （i.p.）, once following injury and once a day for two weeks. MAIN OUTCOME MEASURES： Rats were sacrificed 3, 7, 14, 21, and 28 days after injury, FMN survival after facial nerve injury was analyzed by Toluidine blue staining, and then survival ratios （L/R） were calculated. The number of apoptotic profiles in the injured FMNs were evaluated by TUNEL staining. Expression of Caspase-3 in the facial nucleus was detected by immunohistochemistry methods. RESULTS： A total of 75 rats were included in the final analysi     

Long-term motor cortex reorganization after facial nerve severing in newborn rats

Using the model of facial nerve injury, we have compared the effect of injury in newborn and adult rats on the adult rat motor cortex (M1). To this end, the facial nerve was severed in 10 newborn rats 2 days after birth (Newborn group) and in 10 adult rats (Adult group). In both the Control (contralateral to untouched nerve) and the Experimental (contralateral to severed nerve) hemisphere of each rat, the M1 output organization was assessed by intracortical microstimulation. Our findings demonstrated that: (i) there is no statistical difference in the percentage of movement sites and in current thresholds required to evoke movement in Control hemispheres between the Adult and Newborn groups of rats; (ii) in Adult Experimental hemispheres, neck sites expand in the medial part of the vibrissae representation more extensively than shown in Newborn Experimental hemispheres; (iii) in Newborn Experimental hemispheres eye sites expand in the medial part of the vibrissae representation more extensively than in Adult Experimental hemispheres (these sites overlap the cortical region where electrical stimulation evokes neck movement in Adult Experimental hemispheres) and (iv) in both Newborn and Adult Experimental hemispheres, forelimb sites expand similarly thereby overlapping the same cortical region, corresponding to the lateral part of the vibrissae representation. We conclude that, when the facial nerve injury is performed in the newborn rat, the pattern of movement representation differs from that obtained with the same lesion in the mature brain only in the frontal cortex corresponding to the medial part of the normal vibrissae representation.     

Intracranial stimulation of the facial nerve: normative values with magnetic coil in 240 nerves

This study was aimed at defining normative values of latency and amplitude of the compound muscle action potential (CMAP) from the orbicularis oculi muscle, obtained by transcranial magnetic stimulation. We evaluated the dependence of latency and amplitude on the age of the subjects, in order to calculate cut-off values for each age decade. A total of 120 healthy normal subjects, aged 15–78 years, were studied. CMAP from the orbicularis oculi muscle, pars inferior, was excited by means of transcranial magnetic stimulation of the homolateral scalp. A suitable mathematical model was developed to evaluate the mean variation of the latency and amplitude of CMAP for each age decade in the studied population. Mean values were, respectively, 4.62±0.40 ms for latency and 4.17±0.72 mV for amplitude. We found an increase in latency of about 0.12 ms for every ten years, that became 0.15 ms if referring only to subjects over 20 years, and a decrease in amplitude of 0.18 mV each ten years. Normality interval values for the latency and amplitude were calculated for each age decade. Our data, obtained in a representative population for range of age of evaluated subjects, provide normality values and variability coefficients useful for future confronting works. Received: 20 September 2002 / Accepted in revised form: 6 December 2002 Correspondence to D. Cocito     

Impairment of microglial responses to facial nerve axotomy in cathepsin S-deficient mice

Cathepsin S (CS) is a lysosomal/endosomal cysteine protease especially expressed in cells of a mononuclear lineage including microglia. To better understand the role of CS in microglia, we investigated microglial responses after a facial nerve axotomy in CS-deficient (CS-/-) and wild-type mice. Microglia in both groups accumulated in the facial motor nucleus following axotomy. However, the mean number of microglia in CS-/- mice on the axotomized side was significantly smaller than that in wild-type mice. Microglia were found to adhere to injured motoneurons in wild-type mice, whereas microglia abutted on injured motoneurons without spreading on their surface in CS-/- mice. At the same time, the axotomy-induced down-regulation of tenasin-R, an antiadhesive perineuronal net for microglia, was partially abrogated in CS-/- mice. Primary cultured microglia prepared from CS-/- mice showed that CS deficiency caused significant suppression of migration and transmigration of microglia. In CS-/- mice, impaired recruitments of circulating monocytes and T lymphocytes and reduced expression of the class II major compatibility complex on the axotomized side were observed. Interestingly, cathepsin B, a typical lysosomal cysteine protease, was markedly expressed on the axotomized side in CS-/- but not in wild-type microglia. Finally, we compared axotomy-induced neuronal death in the two groups and found that the percentage of motoneurons that survived in CS-/- mice was significantly smaller than that in wild-type mice. The present study strongly suggests that CS plays a role in the migration and activation of microglia to protect facial motoneurons against axotomy-induced injury.