Constitutive and functional expression of YB-1 in microglial cells.

Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS-treated cultures. YB-1 upregulation was not accompanied by its translocation from the cytoplasm to the nucleus. YB-1 induction appeared to be related to microglial proliferation because it was partially co-regulated with Ki67. In addition, YB-1 protein levels correlated with microglia phagocytic activity because its upregulation could also be induced by inert NPs.

Dysregulation of iron protein expression in the G93A model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons which leads to progressive paralysis and death by respiratory failure. Although the cause of sporadic ALS is still unknown, oxidative stress is suggested to play a major role in the pathogenesis of this disease and of the rare familial form, which often exhibits mutations of the superoxide dismutase 1 (SOD1) gene. Since enhanced iron levels are discussed to participate in oxidative stress and neuronal death, we analyzed the expression levels of Fe-related mRNAs in a cell culture ALS model with the G93A mutation of SOD1. We observed an increased total iron content in G93A-SOD1 SH-SY5Y neuroblastoma cells compared to wild-type (WT)-SOD1 cells. mRNA expression for transferrin receptor 1 (TfR1) and divalent metal transporter 1 was increased in G93A-SOD1 cells, which was in accordance with higher iron uptake. Experiments with the iron chelator deferoxamine revealed a normal reaction of WT and mutant cells to cytoplasmic iron depletion, i.e. TfR1 upregulation, suggesting a basically conserved function of the iron-responsive element/iron regulatory protein (IRE/IRP) pathway, designed to adapt gene expression to iron levels. Expression levels of mitoferrin 1 and 2, frataxin, and iron-sulfur cluster scaffold protein were also significantly increased in G93A-SOD1 cells, suggesting higher mitochondrial iron import and utilization in biosynthetic pathways within the mitochondria. Moreover, expression of these transcripts was further enhanced, if G93A-SOD1 cells were differentiated by retinoic acid (RA). Since RA treatment increased cytoplasmic reactive oxygen species (ROS) levels in these cells, an IRE/IRP independent, ROS-mediated mechanism may account for dysregulation of iron-related genes.

Preconditioning of brain slices against hypoxia induced injury by a Gynostemma pentaphyllum extract--stimulation of anti-oxidative enzyme expression.

A short period of hypoxia/hypoglycaemia (oxygen and glucose deprivation, OGD) induced by perfusion with O(2)/glucose-free medium caused immediate loss and incomplete restoration of evoked field potentials in the CA1 region of transverse hippocampus slices. OGD-dependent decrease in evoked field potentials can be prevented by a proceeding short OGD event (preconditioning). We report about a study investigating the effect of an ethanolic Gynostemma pentaphyllum extract on evoked field potentials when administered before the OGD episode. Using this procedure, the extract completely protected the cells of the slices from functional injury. In an astroglia rich cell culture the ethanolic Gynostemma pentaphyllum extract caused within 48 h of cultivation increased protein and activity levels of the anti-oxidative enzymes manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase (GPx). Consequently, the cellular H(2)O(2) concentration remained at a low level. These data suggest that the Gynostemma pentaphyllum-mediated increase in antioxidative enzyme activities may contribute to the protection of transverse hippocampus slices from OGD induced functional injury. Our results demonstrate that the prophylactic administration of the ethanolic extract from Gynostemma pentaphyllum has a high potential to protect from ischemia/reperfusion injury.

Prevention of free fatty acid-induced lipid accumulation, oxidative stress, and cell death in primary hepatocyte cultures by a Gynostemma pentaphyllum extract.

Hepatocytes of a primary cell culture that are exposed to high glucose, insulin, and linoleic (LA) acid concentration respond with lipid accumulation, oxidative stress up to cell death. Such alterations are typically found in patients with non-alcoholic fatty liver disease (NAFLD). We used this cellular model to study the effect of an ethanolic Gynostemma pentaphyllum (GP) extract in NAFLD. When hepatocytes were cultured in the presence of high insulin, glucose, and LA concentration the extract completely protected the cells from cell death. In parallel, the extract prevented accumulation of triglycerides (TGs) and cholesterol as well as oxidative stress. Our data further demonstrate that GP stimulates the production of nitric oxide (NO) in hepatocytes and affects the molecular composition of the mitochondrial phospholipid cardiolipin (CL). We conclude that GP is able to protect hepatocytes from cell death, lipid accumulation, and oxidative stress caused by diabetic-like metabolism and lipotoxicity. Therefore, GP could be beneficial for patients with diabetes mellitus and NAFLD.

Triggered by asphyxia neurogenesis seems not to be an endogenous repair mechanism, gliogenesis more like it.

We analyzed the long-term consequences of asphyxial cardiac arrest for hippocampal cell proliferation in rats to evaluate if the ischaemia-induced degenerated CA1 region may be repopulated by endogenous (stem) cells. Studies were performed in an asphyxial cardiac arrest model with 5 minutes of asphyxiation and three different survival times: 7, 21, and 90 days. Sham-operated non-asphyxiated rats served as control. Cell proliferation was studied by labeling dividing cells with 5-bromo-2'-deoxy-uridine (BrdU). The neurodegenerative/regenerative pattern at single cell levels was monitored by immunohistochemistry. Alterations of gene expression were analyzed by real-time quantitative RT-PCR. Analysis of BrdU-incorporation demonstrated an increase at 7, 21 as well as 90 days after global ischaemia in the hippocampal CA1 pyramidal cell layer. Similar results were found in the dentate gyrus. Differentiation of BrdU-positive cells, investigated by cell phenotype-specific double fluorescent labeling, showed increased neurogenesis only in the dentate gyrus of animals surviving the cardiac arrest for 7 days. The majority of newcomers, especially in the damaged CA1 region, consisted of glial cells. Moreover, asphyxia seemed to be able to induce the migration of microglia and astroglia from adjacent areas into the damaged area and/or the activation of resident cells. In addition, we show microglia proliferation/activation even 90 days after cardiac arrest. This morphological finding was confirmed by PCR analysis. The results indicate that asphyxia triggers cell proliferation in general and gliogenesis in particular - a possible pro-reparative event. Furthermore, from the finding of microglia proliferation up to 90 days after insult we conclude that delayed cell death processes take place which should be considered for further therapy strategies.

Haloperidol and clozapine decrease S100B release from glial cells.

Recent meta-analyses showed consistently elevated levels of S100B in serum and cerebrospinal fluid of schizophrenic patients. This finding has been attributed to glial pathology because S100B is produced by astrocytes and oligodendrocytes. However, S100B may be likewise associated with schizophrenia-related disturbances in glial cell as well as adipocyte energy supply and glucose metabolism. The influence of antipsychotic drugs on S100B levels remains unclear, and some studies have suggested that treatment with these drugs may actually contribute to the elevated S100B levels observed in schizophrenic patients. In this study, we explored the effects of the typical antipsychotic haloperidol and the atypical prototype drug clozapine on the release of S100B by astrocytic C6 cells and oligodendrocytic OLN-93 cells. Because of the association between schizophrenia and disturbances in energy metabolism, we assessed the effects of these drugs under basal condition (BC) compared to serum and glucose deprivation (SGD). We found that treatment of C6 and OLN-93 cells with haloperidol and clozapine reduced the release of S100B from C6 and OLN-93 cells under BC and SGD in vitro at a tissue concentration corresponding to the assumed therapeutic dose range of these drugs. These data suggest that elevated levels of S100B in bodily fluids of schizophrenic patients are normalized rather than increased by the effects of antipsychotic drugs on glial cells.

Selective induction of apoptosis in glioma tumour cells by a Gynostemma pentaphyllum extract.

At low concentration H(2)O(2) is an important signal molecule in proliferation of tumour cells. We report about a study investigating the effect of an ethanolic extract from Gynostemma pentaphyllum on proliferation of C6 glioma tumour cells and cellular H(2)O(2) concentration. The proliferation of these cells was maximal at about 1 muM extracellular H(2)O(2). HPLC-finger prints of the extract revealed a set of saponines as essential components. In C6 glioma cells the extract caused increase in super oxide dismutase (SOD) activity, in the amount of SOD protein, and in cellular H(2)O(2) concentration. It inhibited cell proliferation and induced activation of caspase 3 as indication of apoptosis. No effect of the extract was observed on the proliferation of astrocytes of a primary cell culture. From these findings we suggest that the ethanolic extract from Gynostemma pentaphyllum may selectively shift the H(2)O(2) concentration to toxic levels exclusively in tumour cells due to increased SOD activity. It may have a high potency in cancer therapy and cancer prophylaxis.

Protection of hippocampal slices against hypoxia/hypoglycemia injury by a Gynostemma pentaphyllum extract.

In transverse hippcampus slices a short period of hypoxia/hypoglycemia induced by perfusion with O(2)/glucose-free medium caused early loss and incomplete restoration of evoked field potentials to only 50% in the CA(1) region. We report about a study investigating the effect of an ethanolic Gynostemma pentaphyllum extract in this system. When given with reperfusion the extract completely protected the cells of the slices from functional injury. The extract also protected at the subcellular level isolated mitochondria which had been subjected to hypoxia/reoxygenation in combination with elevated extramitochondrial Ca(2+) concentration from functional injury. In isolated mitochondria the extract protected from Ca(2+)-induced opening of the mitochondrial permeability transition pore and reduced lipid peroxidation. Our data demonstrate that the ethanolic extract of Gynostemma pentaphyllum has a high potential to protect from ischemia/reperfusion injury. It should be beneficial as prophylactic nutrition supplement and during revascularization of arterial blood vessels from stroke and other ischemic events such as coronary occlusion.

S100B is expressed in, and released from, OLN-93 oligodendrocytes: Influence of serum and glucose deprivation.

S100B (member of a family of proteins that are 100% soluble in ammonium sulfate at neutral pH) has been widely used as astrocyte marker in animal models and in human brain diseases. Recent studies revealed S100B-immunopositivity in oligodendrocytes and O2A oligodendroglial progenitor cells. It is unknown, however, if oligodendrocytes produce S100B themselves, or if the S100B-immunolabeling is caused by binding or absorption of the protein. To address this question, S100B expression and protein release were analyzed in a highly pure oligodendrocytic OLN-93 cell line (from rat), in the astrocytic C6 cell line (from rat) and primary astrocytes. S100B was gene expressed in all cultures, as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. OLN-93 cells and glial fibrillary acidic protein (GFAP)-negative astrocytes expressed the multiligand receptor for advanced glycation end products (RAGE). S100B protein levels were determined in supernatants and cell homogenates by immunoluminometry under normal conditions and after serum and glucose deprivation (SGD). SGD led to a several-fold increased release of S100B (after 6 and 24 h), which was particularly pronounced in primary astrocytes. Increased S100B in cell homogenates was most notable in OLN-93 cells under SGD, indicating activated S100B synthesis. These cells also showed the highest percentage of dead cells, as determined by propidium iodide-positivity, after SGD. Incubation with 0.5, 2 and 5 microg/l exogenous S100B was not toxic to OLN-93 cells. In conclusion, OLN-93 cells produce more S100B under SGD than astrocytes and are more susceptible to cell death upon SGD, which provokes leakage of S100B. Our data indicate active S100B secretion from astrocytes under SGD since highly elevated levels of S100B were detected in the supernatant despite a low percentage of dead cells. The experimental results provide further evidence for a production/release of S100B in/from oligodendrocytes, e.g. in metabolic stress conditions like cerebral ischemia. Studies on S100B in bodily fluids should be carefully interpreted in order to avoid misleading hypotheses concerning the specific involvement of astrocytes, due to the various cellular sources of S100B.

Hypothalamic nitric oxide synthase in affective disorder: focus on the suprachiasmatic nucleus.

Depression is frequently associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which leads to repeated episodes of hypercortisolemia. Hypothalamic paraventricular neurons are believed to trigger these processes by aberrant generation and/or release of corticotropin releasing hormone, oxytocin, vasopressin, and nitric oxide (NO). Recent findings from two independent laboratories have demonstrated that the suprachiasmatic nucleus, which in part controls the cellular activity of paraventricular neurons (PVN), is also involved in affective disorder. The aim of the present study was to elucidate by stereological analysis, whether suprachiasmatic nucleus (SCN) nitric oxide synthase and neurophysin generating neurons are affected in neuropsychiatric disorders. We show that compared to controls the number of nitric oxide synthase immunoreactive neurons is greatly reduced both in depression and in schizophrenia. In subjects with affective disorder there was a correlation between the number of NOS-expressing cells and duration of treatment with antidepressants. The number of neurophysin-expressing SCN neurons was also fewer in cases with mood disorder. It is concluded that SCN-derived NO may be a relevant pathophysiological factor in neuropsychiatric disorders.

Repeated application of ketamine to rats induces changes in the hippocampal expression of parvalbumin, neuronal nitric oxide synthase and cFOS similar to those found in human schizophrenia.

Treatment with the phencyclidine derivative ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist and a well known anesthetic, has recently been introduced to mimic schizophrenia in animals. Using rats repeatedly treated with sub-anesthetic doses we demonstrate in the hippocampal formation the cellular distribution patterns of proteins being relevant to the pathogenesis of schizophrenia. Compared with controls an increase in the density of reduced nicotinamide adenine dinucleotide phosphate diaphorase-, neuronal nitric oxide synthase- and cFOS-positive hippocampal interneurons was found, whereas the density of parvalbumin expressing cells was decreased. Our experiments show that repeated injections of sub-anesthetic doses of ketamine induce significant changes in the nitrergic and GABAergic system which, in part, resemble those described in postmortem brains of human schizophrenics indicating that sub-chronic treatment with sub-anesthetic doses of ketamine might be a useful animal model to study schizophrenia.

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects.

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.

Nitric oxide synthase, an essential factor in peripheral nerve regeneration.

Nitric oxide (NO) exerts both, pro-apoptotic and anti-apoptotic actions and appears to be acritical factor inneuronal degenerative and regenerative processes. NO is synthesized from L-arginine by NO synthase occurring in three isoforms of (neuronal, nNOS; endothelial, eNOS; inducible, iNOS). In a mice sciatic nerve model the regenerative outcome was assessed when the endogenous NO supply was deficient by knocking out the respective NOS isoform and compared to that of wild type mice after nerve transection. In nNOS knock-out mice a delay in regeneration, preceded by slowedWallerian degeneration and a disturbed pruning of uncontrolled sprouts, was observed. This was associated with a delayed recovery of sensory and motor function. Additionally, deficiency of nNOS led after nerve cut to a substantial loss of small and medium-sized dorsal root ganglia neurons, spinal cord interneurons and, to a lesser extent, spinal cord motor neurons. A lack of iNOS resulted in a delayed Wallerian degeneration and impaired regenerative outcome without consequences for neuronal survival. A lack of eNOS was well tolerated, although a delay in nerve revascularization was observed. Thus, after peripheral nerve lesion, regular NOS activity is essential for cell survival and recovery with reference to the nNOS isoform.

[Factors influencing nerve regeneration]. / Faktoren, die die Regeneration peripherer Nerven beeinflussen1.

This paper describes the most important cellular and molecular factors that influence nerve regeneration. The first prerequisite for axonal regeneration is survival of the neuron. This depends on neuron type, age, and the degree and proximity of the injury to the cell body. Spinal motoneurons are less susceptible to injury-induced death than cranial motoneurons and sensory neurons. The surviving neurons undergo changes characteristic of a switch from a transmitting mode to a growing mode. They produce various neurotrophic factors and their receptors influencing the neuron and the non-neuronal cells such as Schwann cells. The distal nerve stump undergoes degenerative processes including removal of axons and phagocytosis of myelin debris, the so-called Wallerian degeneration. Until the second day phagocytosis is done by Schwann cells, hematogenous macrophages invade the distal stump at the second day and phagocyte the whole debris within two weeks. Devoid of axonal contact, the myelinating Schwann cells switch their function from myelination to growth support for the regenerating axons, including cell proliferation, downregulation of myelin components and upregulation of neurotrophic factors. Additionally, the Schwann cells form the so-called Bands of Büngner, cell columns serving as pathway for the growing axon. Trophic factors, cell adhesion molecules and extracellular matrix influence the neuron, the growing axon and the endorgan as well as the non-neuronal cells such as Schwann cells, fibroblasts and macrophages. Application of drugs or trophic substances to enhance nerve regeneration after trauma and reconstruction is in the very beginning, and thus requires further experimental and clinical studies. Experimentally, FK 506 was found to support axonal regeneration after crush lesions and nerve grafting. Growth factors are currently administered clinically in other neurological diseases.

Tissue engineering of peripheral nerves: Epineurial grafts with application of cultured Schwann cells.

After a simple nerve lesion, primary microsurgical suture is the treatment of choice. A nerve gap has to be bridged, with a nerve graft sacrificing a functioning nerve. Alternatively, tissue engineering of nerve grafts has become a subject of experimental research. It is evident that nerve regeneration requires not only an autologous, allogenous, or biodegradable scaffold, but additional interactions with regeneration-promoting Schwann cells. In this study, we compared epineurial and acellularized epineurial tubes with and without application of cultured Schwann cells as alternative grafts in a rat sciatic nerve model. Autologous nerve grafts served as controls. Evaluation was performed after 6 weeks; afterwards, sections of the graft and distal nerve were harvested for histological and morphometrical analysis. Compared to controls, all groups showed a significantly lower number of axons, less well-shaped remyelinizated axons, and a delay in clinical recovery (e.g., toe spread). The presented technique with application of Schwann cells into epineurial tubes did not offer any major advantages for nerve regeneration. Thus, in this applied model, neither the implantation of untreated nor the implantation of acellularized epineurial tubes with cultured Schwann cells to bridge nerve defects was capable of presenting a serious alternative to the present gold standard of conventional nerve grafts for bridging nerve defects in this model.

Further immunohistochemical evidence for impaired NO signaling in the hypothalamus of depressed patients.

The cellular expression of nitric oxide synthase (NOS) was studied in neurons of the Nuc. suprachiasmaticus (SCN) of depressed patients and matched controls. The number of NOS-immunoreactive SCN neurons was significantly reduced in depression. We conclude that affective disorders are accompanied by impaired hypothalamic NO signaling.

[The influence on nerve regeneration by the beta2-receptor agonist clenbuterol]. / Die Beeinflussung der Regeneration peripherer Nerven durch den beta2-Rezeptor-Agonisten Clenbuterol.

PURPOSE: Clenbuterol has shown a neuroprotective action in the central nervous system by induction of growth factors after cellular damage. Additionally, the atrophy of sceletal muscle is attenuated by the application of Clenbuterol after denervation. This experiment was performed to show the influence of Clenbuterol on regeneration of peripheral nerves. MEHOD AND MATERIAL: In a rat model, the sciatic nerve was transected and microsurgically coaptated. Eight animals in each group received a daily oral dosage of 100 microg/kg bodyweight during four, respectively six weeks. Two control groups received only drinking water under the otherwise same protocol. The assessment was done histologically, morphometrically and clinically. The weight ratio of the soleus and extensor digitorum muscles of the operated to the contralateral side was measured. RESULTS: The groups treated with Clenbuterol showed histologically and morphometrically a significantly increased axon count and a better g-ratio. The muscle weight ratio was significantly higher in the Clenbuterol group after six weeks and more animals in this group were able to spread their toes. CONCLUSION: The oral application of Clenbuterol in a rodent model shows a positive influence on nerve regeneration.

Strain specific differences in a cardio-pulmonary resuscitation rat model.

An asphyxial cardiac arrest rat model, originally developed for Sprague-Dawley rats, was transferred to a Wistar rat model. Several strain specific life support adjustments, i.e. ventilator settings, anaesthesia, and drug requirements, were necessary to stabilize the model for Wistar rats. Despite these arrangements numerous resuscitation related variables appeared different. Three groups were evaluated and compared: a temperature monitored Wistar group 1 (n=34), a temperature controlled Wistar group 2 (n=26) and a temperature controlled Sprague-Dawley group 3 (n=7). Overall, Wistar rats seem to have more sensitive cardio-circulatory system evidenced by a more rapid development of cardiac arrest (164 vs. 201 s), requiring higher adrenaline/epinephrine doses (10 vs. 5 microg/kg) and requiring more time for recovery after resuscitation (i.e. for return of blood pressure and blood gases). Without strict temperature control (as in groups 2+3 rats) group 1 rats went into spontaneous mild to moderate hypothermia during the first 24 h after restoration of spontaneous circulation (ROSC). Spontaneous hypothermia delayed the development of overall visible CA1 neuronal damage 24-48 h, but did not prevent it; therefore the model seemed to be suitable for future studies. Neuronal damages in the CA1 region in Wistar rats appeared to be more as shrunken cell bodies and pyknotic nuclei before resorption took place, whereas in Sprague-Dawley rats appeared in the same region.

Regeneration of peripheral nerves after clenbuterol treatment in a rat model.

Clenbuterol is known to act as a neuroprotective substance in the central nervous system, and also reduces muscle atrophy after denervation. The aim of this study was to evaluate its influence on peripheral nerve regeneration. The rat sciatic nerve model was used in four groups (n = 8 per group). After complete nerve transection and microsurgical coaptation, two groups received a daily oral dose of 100 microg/kg clenbuterol and two served as controls. Regeneration was assessed clinically, histologically, and morphometrically after 4 and 6 weeks. The weight ratios of calf muscles were calculated. Histological examination showed significantly increased axon counts in the clenbuterol group and a better degree of myelination. Muscle weight ratios of the clenbuterol group were significantly increased after 6 weeks, and the animals showed improved function of the hindlimb. Thus, therapy with 100 microg/kg clenbuterol daily after coaptation of a sciatic nerve showed a positive influence on clinical, histological, and morphometrical parameters in the rat model. The underlying mechanism remains unclear.