Phosphorylation of extracellular signal-regulated kinase 1/2 in subepidermal nerve fibers mediates hyperalgesia following diabetic peripheral neuropathy.

Peripheral neuropathy, a chronic complication of diabetes mellitus (DM), is often accompanied by the onset of severe pain symptoms that affect quality of life. However, the underlying mechanisms remain elusive. In the present study, we used Sprague-Dawley rats to establish a rodent model of the human type 1 DM by a single intraperitoneal (i.p.) injection with streptozotocin (STZ) (60 mg/kg). Hypersensitivity, including hyperalgesia and allodynia, developed in the STZ-induced diabetic rats. Cutaneous innervation exhibited STZ-induced reductions of protein gene product 9.5-, peripherin-, and neurofilament 200-immunoreactivity (IR) subepidermal nerve fibers (SENFs). Moreover, the decreases of substance P (SP)- and calcitonin gene-related peptide (CGRP)-IR SENFs were distinct gathered from the results of extracellular signal-regulated kinase 1 and 2 (ERK1/2)- and phosphorylated ERK1/2 (pERK1/2)-IR SENFs in STZ-induced diabetic rats. Double immunofluorescence studies demonstrated that STZ-induced pERK1/2-IR was largely increased in SENFs where only a small portion was colocalized with SP- or CGRP-IR. By an intraplantar (i. pl.) injection with a MEK inhibitor, U0126 (1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), hyperalgesia was attenuated in a dose-responsive manner. Botulinum toxin serotype A had dose-dependent analgesic effects on STZ-induced hyperalgesia and allodynia, which exhibited equivalent results as the efficacy of transient receptor potential vanilloid (TRPV) channel antagonists. Morphological evidence further confirmed that STZ-induced SP-, CGRP- and pERK1/2-IR were reduced in SENFs after pharmacological interventions. From the results obtained in this study, it is suggested that increases of pERK1/2 in SENFs may participate in the modulation of TRPV channel-mediated neurogenic inflammation that triggers hyperalgesia in STZ-induced diabetic rats. Therefore, ERK1/2 provides a potential therapeutic target and efficient pharmacological strategies to address hyperglycemia-induced neurotoxicity.

Nerve Decompression Improves Spinal Synaptic Plasticity of Opioid Receptors for Pain Relief.

Nerve decompression is an essential therapeutic strategy for pain relief clinically; however, its potential mechanism remains poorly understood. Opioid analgesics acting on opioid receptors (OR) within the various regions of the nervous system have been used widely for pain management. We therefore hypothesized that nerve decompression in a neuropathic pain model of chronic constriction injury (CCI) improves the synaptic OR plasticity in the dorsal horn, which is in response to alleviate pain hypersensitivity. After CCI, the Sprague-Dawley rats were assigned into Decompression group, in which the ligatures around the sciatic nerve were removed at post-operative week 4 (POW 4), and a CCI group, in which the ligatures remained. Pain hypersensitivity, including thermal hyperalgesia and mechanical allodynia, was entirely normalized in Decompression group within the following 4 weeks. Substantial reversal of mu- and delta-OR immunoreactive (IR) expressions in Decompression group was detected in primary afferent terminals in the dorsal horn. In Decompression group, mu-OR antagonist (CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 [Disulfide Bridge: 2-7]) and delta-OR antagonist (NTI, 17-(cyclopropylmethyl)-6,7-dehydro-4,5α-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan hydrochloride) re-induced pain hypersensitivity by intrathecal administration in a dose-responsive manner. Additionally, mu-OR agonist (DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin) and delta-OR agonist (SNC80, ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide) were administrated intrathecally to attenuating CCI-induced chronic and acute pain hypersensitivity dose-dependently. Our current results strongly suggested that nerve decompression provides the opportunity for improving the synaptic OR plasticity in the dorsal horn and pharmacological blockade presents a novel insight into the therapeutic strategy for pain hypersensitivity.

Intact subepidermal nerve fibers mediate mechanical hypersensitivity via the activation of protein kinase C gamma in spared nerve injury.

BACKGROUND: Spared nerve injury is an important neuropathic pain model for investigating the role of intact primary afferents in the skin on pain hypersensitivity. However, potential cellular mechanisms remain poorly understood. In phosphoinositide-3 kinase pathway, pyruvate dehydrogenase kinase 1 (PDK1) participates in the regulation of neuronal plasticity for central sensitization. The downstream cascades of PDK1 include: (1) protein kinase C gamma (PKCg) controls the trafficking and phosphorylation of ionotropic glutamate receptor; (2) protein kinase B (Akt)/the mammalian target of rapamycin (mTOR) signaling is responsible for local protein synthesis. Under these statements, we therefore hypothesized that an increase of PKCg activation and mTOR-dependent PKCg synthesis in intact primary afferents after SNI might contribute to pain hypersensitivity. RESULTS: The variants of spared nerve injury were performed in Sprague-Dawley rats by transecting any two of the three branches of the sciatic nerve, leaving only one branch intact. Following SNIt (spared tibial branch), mechanical hyperalgesia and mechanical allodynia, but not thermal hyperalgesia, were significantly induced. In the first footpad, normal epidermal innervations were verified by the protein gene product 9.5 (PGP9.5)- and growth-associated protein 43 (GAP43)-immunoreactive (IR) intraepidermal nerve fibers (IENFs) densities. Furthermore, the rapid increases of phospho-PKCg- and phosphomTOR-IR subepidermal nerve fibers (SENFs) areas were distinct gathered from the results of PGP9.5-, GAP43-, and neurofilament 200 (NF200)-IR SENFs areas. The efficacy of PKC inhibitor (GF 109203X) or mTOR complex 1 inhibitor (rapamycin) for attenuating mechanical hyperalgesia and mechanical allodynia by intraplantar injection was dose-dependent. CONCLUSIONS: From results obtained in this study, we strongly recommend that the intact SENFs persistently increase PKCg activation and mTOR-dependent PKCg synthesis participate in the initiation and maintenance of mechanical hypersensitivity in spared nerve injury, which represents as a novel insight into the therapeutic strategy of pain in the periphery.

Scratch wound closure of myoblasts and myotubes is reduced by inflammatory mediators.

Complex interactions exist between muscle repair processes and acute inflammatory responses that are initiated by exercise-induced muscle damage. The purpose of this study was to examine whether inflammatory mediators secreted by activated macrophages affect the migration of myogenic cells to the injury site. Migration was measured using a scratch wound closure assay in C2 C12 -derived myogenic cells incubated in activated macrophage-conditioned medium. Both myoblast and myotube migrations were significantly reduced in activated macrophage-conditioned medium compared with control medium. Furthermore, we demonstrated that the inhibitory effect on myoblast and myotube migrations was mediated, at least in part, by the two major cytokines secreted by activated macrophages, tumour necrosis factor (TNF)-α and interleukin (IL)-6. These findings suggest that the migration rate of myogenic cells may be reduced by inflammatory mediators. It may provide useful insights for future researches on the role of macrophages in the process of muscle repair and regeneration.

Salivary Immuno Factors, Cortisol and Testosterone Responses in Athletes of a Competitive 5,000 m Race.

The exercise-stress model can be a model of temporary immunosuppression that occurs after severe physical and psychological stress. It also allows for the study of interactions between the endocrine and the immune systems. This study examined changes in salivary hormonal and immune factors in athletes in response to physical and psychological stress in a 5,000 m running competition. Eighteen endurance-trained runners (9 males and 9 females) participated in this study. All participants completed a competitive 5,000 m race. Saliva samples were collected 10 min before (PRE) and 10 min after (POST) the competition. Saliva was analyzed for α-amylase activity, concentrations of salivary immunoglobulin A (SIgA), lactoferrin, cortisol, testosterone and total protein. Although the concentrations of salivary TP, SIgA, lactoferrin, cortisol and α-amylase activity were significantly increased immediately after a competitive 5,000 m race, the secretion rates of these factors were not significantly altered in both male and female groups. Additionally, basal levels of SIgA and α-amylase activity were significantly higher in female runners than in male runners. This gender difference still existed after the race. The secretion rates of testosterone decreased significantly after the race in the male, but not in the female group. Moreover, testosterone-to-cortisol (T/C) ratios were significantly lower post-competition compared to pre-competition in both male and female athletes. The T/C ratio had been used as a performance index for athletes. Whether there are correlations between these changes of their physiological characteristics and better running performance need further investigations.

Nerve demyelination increases metabotropic glutamate receptor subtype 5 expression in peripheral painful mononeuropathy.

Wallerian degeneration or nerve demyelination, arising from spinal nerve compression, is thought to bring on chronic neuropathic pain. The widely distributed metabotropic glutamate receptor subtype 5 (mGluR5) is involved in modulating nociceptive transmission. The purpose of this study was to investigate the potential effects of mGluR5 on peripheral hypersensitivities after chronic constriction injury (CCI). Sprague-Dawley rats were operated on with four loose ligatures around the sciatic nerve to induce thermal hyperalgesia and mechanical allodynia. Primary afferents in dermis after CCI exhibited progressive decreases, defined as partial cutaneous denervation; importantly, mGluR5 expressions in primary afferents were statistically increased. CCI-induced neuropathic pain behaviors through the intraplantar injections of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, were dose-dependently attenuated. Furthermore, the most increased mGluR5 expressions in primary afferents surrounded by reactive Schwann cells were observed at the distal CCI stumps of sciatic nerves. In conclusion, these results suggest that nerve demyelination results in the increases of mGluR5 expression in injured primary afferents after CCI; and further suggest that mGluR5 represents a main therapeutic target in developing pharmacological strategies to prevent peripheral hypersensitivities.

A high-efficiency induction of dopaminergic cells from human umbilical mesenchymal stem cells for the treatment of hemiparkinsonian rats.

The success rate in previous attempts at transforming human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton's jelly of the umbilical cord into dopaminergic cells was a mere 12.7%. The present study was therefore initiated to establish a more effective procedure for better yield of dopaminergic cells in such transformation for more effective HUMSC-based therapy for parkinsonism. To examine, in vitro, the effects of enhanced Nurr1 expression in HUMSCs on their differentiation, cells were processed through the three-stage differentiation protocol. The capacity of such cells to synthesize and release dopamine was measured by HPLC. The therapeutic effects of Nurr1-overexppressed HUMSCs were examined in 6-hydroxydopamine-lesioned rats by quantification of rotations in response to amphetamine. Enhanced Nurr1 expression in HUMSCs promoted the transformation into dopaminergic cells in vitro through stepwise culturing in sonic hedgehog, fibroblast growth factor-8, and neuron-conditioned medium. The success rate was about 71%, as determined by immunostaining for tyrosine hydroxylase and around 94 nM dopamine synthesis (intracellular and released into the culture medium), as measured by HPLC. Additionally, transplantation of such cells into the striatum of hemiparkinsonian rats resulted in improvement of their behavioral deficits, as indicated by amphetamine-evoked rotation scores. Viability of the transplanted cells lasted for at least 3 months as verified by positive staining for tyrosine hydroxylase. Nurr1, FGF8, Shh, and NCM can synergistically enhance the differentiation of HUMSCs into dopaminergic cells and may pave the way for HUMSC-based treatments for Parkinson's disease.

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, "Green King". Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

Redistribution of voltage-gated sodium channels after nerve decompression contributes to relieve neuropathic pain in chronic constriction injury.

Nerve decompression is an important therapeutic strategy to relieve neuropathic pain and promote the peripheral nerve regeneration. To address these issues, we investigated the effects of nerve decompression on relief of neuropathic pain behaviors, redistribution of voltage-gated sodium channels (VGSCs), and skin reinnervation with chronic constriction injury (CCI). At post-operative week (POW) 4, animals were divided into a decompression group, in which the ligatures were removed, and a CCI group, in which the ligatures remained. Thermal hyperalgesia and mechanical allodynia at POW 8 had distinct reductions in decompression group compared to CCI group. At that time in CCI group, morphological evidence of pan VGSCs (Pan Nav) and isoforms of VGSCs (Nav1.6, Nav1.9, except for Nav1.8) were shown the widely distribution along the injured sciatic nerve. All of the VGSCs in decompression group became clustering around the node of Ranvier, similar to the pattern of control sciatic nerve at POW 8. Skin reinnervation was demonstrated by epidermal nerve density (END) for protein gene product 9.5 (PGP 9.5)-immunoreactive (IR) nerve fibers and a significant difference between groups only at POW 24 (p=0.01). Growth-associated protein 43 (GAP-43) is participated in the nerve fiber growth and sprouting, a difference in END for GAP-43-IR nerve fibers at POW 24 between groups were also significant (p=0.02). These observations demonstrated that nerve decompression was accompanied with the disappearance of neuropathic pain behaviors after CCI. Morphological studies provided the evidence that redistribution of VGSCs along the injured sciatic nerve but still with an incomplete skin reinnervation. These significant findings demonstrated a role of VGSCs in the pathogenesis of neuropathic pain, and gave an approaching in pharmacological basis of therapeutics.

Peptidergic intraepidermal nerve fibers in the skin contribute to the neuropathic pain in paclitaxel-induced peripheral neuropathy.

Paclitaxel in chemotherapy-induced peripheral neuropathy (CIPN) is predominantly with a dose-limiting effect on neuropathic pain in clinical strategy. In the present study, the relationship between the neuropathic pain and nerve degeneration in paclitaxel CIPN was investigated. Adult male Sprague-Dawley (SD) rats were divided into three paclitaxel groups (0.5, 1.0, 2.0mg/kg) and a vehicle group with four intraperitoneal (i.p.) injections on alternating days. Our results demonstrated that the paclitaxel groups significantly exhibited the reductions of thermal hyperalgesia and mechanical allodynia. The neurotoxicity of paclitaxel conveyed the degeneration of intraepidermal nerve fibers (IENFs) in hindpaw glabrous skin. Nevertheless, the influence of paclitaxel to the peptidergic IENFs are even unknown. The skin innervation of protein gene product 9.5 (PGP 9.5)-immunoreactive (IR) IENFs in paclitaxel groups revealed the decreasing levels of density (73.54±0.72%, 63.17±1.77%, 61.79±2.68%, respectively; vs. vehicle group, p<0.05) throughout the entire experimental period. Additionally, the diminishing levels of density for peptidergic substance P (SP)-IR IENFs in paclitaxel groups were significantly shown (48.84±1.74%, 30.02±1.69%, 30.14±0.37%, respectively; vs. vehicle group, p<0.05). On the contrary, the density for peptidergic calcitonin gene-related peptide (CGRP)-IR IENFs in paclitaxel groups were revealed the similar decreasing levels (82.75±0.91%, 84.34±3.20%, 81.99±0.25%, respectively; vs. vehicle group, p<0.05). Linear regression analyses exhibited that densities of IENFs for PGP 9.5, SP, CGRP were correlated with withdrawal latencies (r(2)=0.77, p<0.0001; r(2)=0.75, p<0.0001; r(2)=0.28, p=0.0001, respectively) and mechanical thresholds (r(2)=0.43, p<0.0001; r(2)=0.73, p<0.0001; r(2)=0.40, p<0.0001, respectively). Therefore, the present results suggested that the development of neuropathic pain following paclitaxel injection induced the progressive degeneration of IENFs in skin and gave the evidence that the peptidergic IENFs may play an important role in therapeutic strategy of paclitaxe CIPN.

Steady-state pharmacokinetics and tissue distribution of anthraquinones of Rhei Rhizoma in rats.

AIM OF THE STUDY: Rhei Rhizoma, the rhizome of Rheum palmatum L. (RP), is a popular herb in clinical Chinese medicine. RP is abundant in polyphenolic anthraquinones, which have been reported to show various beneficial bioactivities. This study investigated the pharmacokinetics and tissue distribution of anthraquinones following seven-dose administration of RP decoction to rats. MATERIALS AND METHODS: Six Sprague-Dawley rats were given 2.0 g/kg of RP twice daily for seven doses and blood samples were collected at designated time after the 7th dose. Another six rats were sacrificed at 30 min after the 7th dose and organs including liver, kidney, lung and brain were collected. Serum and tissue specimens were assayed by HPLC before and after hydrolysis with ß-glucuronidase and sulfatase, respectively. RESULTS: Pharmacokinetic analysis indicated that the anthraquinones in serum mainly presented as glucuronides/sulfates and contained higher ratio of sulfates when compared with single-dose administration of RP. Contrary to the finding in serum, tissue analysis discovered mainly free form of anthraquinone in most organs assayed, such as aloe-emodin and rhein in kidney, liver, lung; emodin in liver, lung; trace of chrysophanol in kidney and liver. In all brains, neither free forms nor their glucuronides/sulfates have been detected. CONCLUSIONS: The glucuronides/sulfates of anthraquinones were the major forms in bloodstream, whereas the free forms of most anthraquinones were predominant in kidney and liver.

Flavonoid pharmacokinetics and tissue distribution after repeated dosing of the roots of Scutellaria baicalensis in rats.

Scutellariae Radix (root of Scutellaria baicalensis, SR) contains numerous flavonoids such as baicalin, baicalein, and wogonin. This study investigated the pharmacokinetics and tissue distribution of flavonoids and their metabolites in rats after repeated dosing of a SR decoction. Sprague-Dawley rats were orally administered SR at 2 g/kg for seven doses. After the 7th dose, blood samples were withdrawn at specific times and organs, including the liver, kidney, lung, and brain, and collected. The concentrations of baicalein and wogonin in the serum and various tissues were assayed by HPLC before and after hydrolysis with glucuronidase and sulfatase. Baicalein and wogonin were not detected in the serum, and the molecules found were their glucuronides/sulfates. In tissues, the free forms of baicalein and wogonin appeared in the liver, kidney, and lung in addition to their glucuronides/sulfates. Baicalein was the major form in the lung, whereas baicalein glucuronides/sulfates were the major forms in the liver and kidney. Wogonin was the major form in the liver, kidney, lung, and traces of wogonin glucuronides/sulfates were detected in the kidney and liver. Neither baicalein and wogonin nor their glucuronides/sulfates were detected in the brain. In conclusion, the glucuronides/sulfates of baicalein and wogonin were exclusively present in the circulation, whereas their free forms appeared in the lung, liver, and kidney.

Relationships among salivary immunoglobulin A, lactoferrin and cortisol in basketball players during a basketball season.

The aim of this study was to examine the changes and relationships of immune and stress parameters of basketball players during a basketball season. Eight members of National Taichung University basketball team volunteered to participate. Saliva samples were collected at rest and before the start of practice or competition at seven time points during the intense training, competition and recovery period. Salivary immunoglobulin A (sIgA), cortisol, and lactoferrin were measured during training and competition period and compared with those measured at the fourth recovery week. Relationships among immune and stress parameters were evaluated. Compared with those detected at the fourth recovery week, significant decreases in secretion rates and absolute concentrations of sIgA and lactoferrin were observed at times of intense training and competition. In addition, significant increases in secretion rates and absolute concentrations of salivary cortisol were observed during intense training and competition period and the first week of recovery. Moreover, a significant inverse correlation (r = -0.28; P < 0.05) that existed between secretion rates of sIgA and cortisol as well as a positive correlation (r = 0.32; P < 0.05) that existed between secretion rates of sIgA and lactoferrin was measured. Our results demonstrated that the secreted cortisol was induced and the mucosal immunity of the participants was suppressed during the basketball season. The inverse correlation existed between secretion rates of sIgA and cortisol may indicate a possible role of cortisol in the strenuous exercise-induced immunosuppression. Our results also suggest that a delicate balance may exist between mucosal innate and adaptive immune responses.

The interactive effect of exercise and immunosuppressant cyclosporin A on immune function in mice.

Cyclosporin A (CsA) is an effective immunosuppressive agent and exerts its actions by interfering with the activation of T cells. There is growing evidence that regular exercise improves immune function. However, the effects of exercise on immune functions in patients taking CsA are unclear. Here, we examine the interactive effects of CsA administration and regular exercise on immune function in mice. Forty-eight BALB/c mice were randomly assigned to one of six groups with eight mice per group: 0-Ex (no CsA/no exercise), 0 + Ex (no CsA + exercise), 10-Ex (10 mg kg(-1) day(-1) CsA/no exercise), 10 + Ex (10 mg kg(-1) day(-1) CsA + exercise), 20-Ex (20 mg kg(-1) day(-1) CsA/no exercise), and 20 + Ex (20 mg kg(-1) day(-1) CsA + exercise). The three exercise groups were trained for 8 weeks, three times a week, at approximately 75% maximum oxygen uptake (VO(2max)). Nitric oxide and interferon-gamma secretions by mitogen-activated macrophages and spleen cells, respectively, were higher in exercise groups than in non-exercise groups receiving the same doses of CsA. The results of this study indicate that regular exercise may enhance Type I helper T cell functions in mice receiving 10 or 20 mg kg(-1) day(-1) CsA. Our results demonstrate that moderate regular exercise modulates the immune function of CsA-treated mice. However, whether this exercise-induced immunomodulatory effect is beneficial or detrimental to CsA-treated patients needs to be clarified.

Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord.

BACKGROUND: Human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton's jelly of the umbilical cord can be easily obtained and processed compared with embryonic or bone marrow stem cells. These cells may be a valuable source in the repair of spinal cord injury. METHODOLOGY/PRINCIPAL FINDINGS: We examine the effects of HUMSC transplantation after complete spinal cord transection in rats. Approximately 5x10(5) HUMSCs were transplanted into the lesion site. Three groups of rats were implanted with either untreated HUMSCs (referred to as the stem cell group), or HUMSCs treated with neuronal conditioned medium (NCM) for either three days or six days (referred to as NCM-3 and NCM-6 days, respectively). The control group received no HUMSCs in the transected spinal cord. Three weeks after transplantation, significant improvements in locomotion were observed in all the three groups receiving HUMSCs (stem cell, NCM-3 and NCM-6 days groups). This recovery was accompanied by increased numbers of regenerated axons in the corticospinal tract and neurofilament-positive fibers around the lesion site. There were fewer microglia and reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the stem cell group than in the control group. Transplanted HUMSCs survived for 16 weeks and produced large amounts of human neutrophil-activating protein-2, neurotrophin-3, basic fibroblast growth factor, glucocorticoid induced tumor necrosis factor receptor, and vascular endothelial growth factor receptor 3 in the host spinal cord, which may help spinal cord repair. CONCLUSIONS/SIGNIFICANCE: Transplantation of HUMSCs is beneficial to wound healing after spinal cord injury in rats.

Expression of protein gene product 9.5, tyrosine hydroxylase and serotonin in the pineal gland of rats with streptozotocin-induced diabetes.

Hyperglycemia is a well-known factor in reducing nocturnal pineal melatonin production. However, the mechanism underlying diabetes-induced insufficiency of pineal melatonin has remained uncertain. This study was undertaken to examine the structure, innervation and functional activity of the pineal gland in streptozotocin (STZ)-induced diabetes in rats by immunohistochemistry, Western blotting and image analysis. The number of the pinealocytes and the volume of pineal were also estimated using stereologic quantification including the optical fractionator and Cavalieri's method. It has also shown a progressive reduction of the total area of the pineal gland and the nuclear size of pinealocytes beginning at 4 weeks of induced diabetes. Surprisingly, the immunoreactive intensities and protein amounts of serotonin (5-HT) and protein gene product (PGP) 9.5 in the pineal gland were progressively increased from 4 weeks of diabetes. Meanwhile, nerve fibers immunoreactive for PGP 9.5 had disappeared. Diabetes-induced neuropathy was observed in nerve fibers containing tyrosine hydroxylase (TH). The affected nerve fibers appeared swollen and smooth in outline but they showed a distribution pattern, packing density and protein levels comparable to those of the age-matched control animals. Ultrastructural observations have revealed diabetes-induced deformity of Schwann cells and basal lamina, accumulation of synaptic vesicles and deprivation of the dense-core vesicles in the axon terminals and varicosities. The increase in immunoreactivities in 5-HT and PGP 9.5 and shrinkage of pineal gland in the diabetic rats suggest an inefficient enzyme activity of the pinealocytes. This coupled with the occurrence of anomalous TH nerve fibers, may lead to an ineffective sympathetic innervation of the pinealocytes resulting in reduced melatonin production in STZ-induced diabetes.

Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

Human mesenchymal stem cells isolated from Wharton's jelly of the umbilical cord were induced to transform into dopaminergic neurons in vitro through stepwise culturing in neuron-conditioned medium, sonic hedgehog, and FGF8. The success rate was 12.7%, as characterized by positive staining for tyrosine hydroxylase (TH), the rate-limiting catecholaminergic synthesizing enzyme, and dopamine being released into the culture medium. Transplantation of such cells into the striatum of rats previously made Parkinsonian by unilateral striatal lesioning with the dopaminergic neurotoxin 6-hydroxydopamine partially corrected the lesion-induced amphetamine-evoked rotation. Viability of the transplanted cells at least 4 months after transplantation was identified by positive TH staining and migration of 1.4 mm both rostrally and caudally. These results suggest that human umbilical mesenchymal stem cells have the potential for treatment of Parkinson's disease.

GABAergic neuron death in the striatum following kainate-induced damage of hippocampal neurons: evidence for the role of NO in locomotion.

The authors examined the role of nitric oxide (NO) in the relationship between kainate-induced neuronal death and locomotion changes. Locomotion was significantly increased 1 h after kainate injection, suggesting that kainate induced NO and dopamine release. Cell death occurred in the CA1 (41%) and CA3 (54%) regions at 12 h. At 7 days, GABAergic neurons in striatum were lost, suggesting possible pyramidal neuron synapse with striatal GABAergic neurons, and pyramidal neuron damage leading to deafferentation and degeneration of striatal GABAergic neurons. Pre-administration of Nw-nitro-L-arginine-methyl-ester or 7-nitroindazole reduced these effects. These results indicate that NO may modulate kainate-induced neuronal death and locomotion.

Neuropathology of skin denervation in acrylamide-induced neuropathy.

Previous studies have established the neurotoxicity and pathology of acrylamide to large-diameter nerves. It remains unclear (1) whether small-diameter sensory nerves are vulnerable to acrylamide and (2) if so, how the pathology evolves during intoxication. We investigated the influence of acrylamide on small-diameter sensory nerves by studying the pathology of sensory nerve terminals in the skin. The neurotoxic effects of acrylamide (400 ppm in drinking water) on mice were assessed by immunostaining the skin with protein gene product 9.5, a ubiquitin C-terminal hydrolase, particularly useful for demonstrating cutaneous nerve terminals. Within 5 days of acrylamide administration (the initial stage), epidermal nerves showed two major changes: (1) terminal swelling and (2) increased branching. There was a progressive reduction in epidermal nerve density (END) thereafter. Fifteen days after acrylamide intoxication (the late stage), reduction in END became evident (25.22 +/- 2.19 fibers/mm vs 41.74 +/- 2.60 fibers/mm in control mice, P < 0.003). At this stage, there was significant dermal nerve degeneration with ultrastructural demonstrations of vacuolar changes. These findings establish the pathological consequences of acrylamide neurotoxicity in cutaneous sensory nerves with far-reaching implications: (1) providing an animal system to study "dying-back" pathology of nociceptive nerves and (2) forming the ultrastructural foundation for interpreting the pathology of cutaneous nerve degeneration in skin biopsies.