Ganglioside Treatment Modifies Abnormal Elemental Composition in Peripheral Nerve Myelinated Axons of Experimentally Diabetic Rats

Abstract: Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na⁺,K⁺-ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Cholinergic Neuronotrophic Factors. Concurrent Activities on Certain Nerve Growth Factor-Responsive Neurons

Abstract: We had previously reported that in vitro survival of chick embryo ciliary ganglionic neurons can be assured by the addition to the culture medium of appropriate amounts of soluble macromolecular agents termed ciliary neuronotrophic factors. Particularly rich sources of one such factor are aqueous extracts from chick embryo intraocular tissues that include the smooth and striated musculature innervated by ciliary ganglionic neurons. We report here that this eye extract also contains agents that we term ganglionic neuronotrophic factors that support the survival of 11-day chick embryo sympathetic and neonatal mouse dorsal root ganglionic neurons, two traditional targets of nerve growth factor (NGF). Using a recently developed microassay procedure we found that these ganglionic activities are not inactivated by rabbit, rat, or guinea pig antisera raised against the 2.5S (beta) subunit of male mouse submaxillary NGF, rabbit antisera against 7S NGF, or quail antisera against cobra venom NGF. Both the ciliary and ganglionic activities can be quantitated simultaneously by using 24-h in vitro microassays, thus permitting a direct comparison of their respective properties. Both activities were found to (a) adsorb to DE52 cellulose and coelute at a similar salt concentration, (b) focus and be recovered from isoelectric polyacrylamide gels at exactly the same pH region, (c) be heat-and partially acid-labile, but base-stable, and (d) be inactivated by exposure to trypsin. These results suggest that the ciliary and ganglionic neuronotrophic activities are associated with the same protein.     

Effects of Bone Marrow Stromal Cell-conditioned Medium on Primary Cultures of Peripheral Nerve Tissues and Cells

Implantation of bone marrow stromal cells (MSCs) produces an improved functional outcome of peripheral nerve repair. In this study, rat dorsal root ganglion (DRG) explants, rat DRG neurons, and rat Schwann cells (SCs) were treated with monkey MSC-conditioned medium, respectively, and then subjected to MTT assay, Bromodeoxyuridine/Hoechst 33342 double staining, flow cytometry, immunohistochemistry, real-time quantitative PCR, and Western blot analysis, respectively. The results showed that MSC-conditioned medium enhanced axon growth and neurogenesis in cultured DRG explants, augmented cell survival of and expression of NF and GAP-43 by cultured DRG neurons, promoted cell survival and proliferation of cultured SCs, and increased the expression of NGF, BDNF, and bFGF in cultured SCs. We also found that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 1/2 pathway was involved in the enhanced cell proliferation of SCs evoked by MSC-conditioned medium. The data of this study might help the understanding of MSCs-based treatment for peripheral nerve repair.     

大鼠坐骨神经切断后谷氨酰胺合成酶在DRG内的表达变化

目的:观察坐骨神经切断后不同时间点背根神经节(DRG)内谷氨酰胺转化酶(GS)的表达变化。方法:48只SD大鼠随机分为实验组和正常对照组,其中实验组左侧为对照侧,右侧行坐骨神经切断。实验组大鼠分别存活1、3、7、14或21天。免疫组化方法检测DRG中GS的表达。结果:正常组DRG内GS主要表达于卫星细胞。坐骨神经切断1天后GS表达增加,明显高于正常组(P<0.05),3天时GS表达下降,7d时恢复正常。14天、21天时GS表达继续下降,明显低于正常组(P<0.05)。实验组手术侧和对照侧GS表达无显著性差异(P>0.05)。结论:坐骨神经切断后DRG内GS表达存在时空变化,这可能与坐骨神经切断后DRG内谷氨酸介导的兴奋性毒性有关。     

超声微泡介导hVEGF165 基因靶向转染糖尿病鼠缺血骨骼肌

目的：探讨超声介导微泡破裂法促进血管内皮生长因子（VEGF）基因在糖尿病鼠缺血骨骼肌内转染的作用,评估其转染效 率和安全性。方法：建立糖尿病鼠缺血骨骼肌动物模型,以绿色荧光蛋白基因为报告基因, 观察接受超声及微泡治疗组hVEGF165 基因在糖尿病鼠缺血骨骼肌内表达,并与对照组相比。同时取糖尿病鼠缺血骨骼肌进行HE染色行组织学检查。结果：在超声介导 微泡破裂组内,hVEGF165 基因表达明显增强(42.87± 5.12),与单纯接受质粒治疗组(5.02± 1.21)和接受质粒和超声治疗组（8.16± 2.43）相比,差异具有统计学意义（P<0.001）,HE 切片未发现肌组织结构的改变。结论：超声介导微泡破裂法能有效促进外源基因 在糖尿病鼠缺血骨骼肌中表达, 为糖尿病周围血管疾病的基因治疗提供了实验依据。     

福尔马林致痛对大鼠降钙素基因相关肽的影响

目的:研究福尔马林致痛后大鼠脊髓和背根神经节(dorsal root ganglion,DRG)内降钙素基因相关肽(calcitonin gene-related peptide,CGRP)的时空变化规律,为探讨CGRP在伤害性信息传递中的机制和作用提供实验依据。方法:选取健康成年正常SD大鼠54只随机分为生理盐水对照组和福尔马林实验组;实验组为右侧足底皮下给予0.1 mL 5%福尔马林后,分别存活15 min、30min、1 h、3 h、6 h、12h、24h和72h(n=6),免疫组化结合图像分析技术观测CGRP在脊髓腰段及L4~6DRG的表达变化。结果:正常DRG、脊髓前角和后角内CGRP有一定的基础表达。福尔马林致痛后3 h DRG的CGRP表达上升,12 h~24 h达峰值,72 h基本降低到正常;福尔马林致痛后6 h脊髓后角CGRP的表达上调,24 h达到高峰,72 h降低至正常;脊髓前角CGRP未见明显变化。结论:福尔马林致痛引起DRG和脊髓的CGRP的表达呈现一定的时空模式,可能是其参与伤害性信息传递的机制之一。     

天麻素通过下调Nav1.6通道表达缓解糖尿病神经病理性疼痛的研究

目的:探究天麻素对Ⅱ型糖尿病神经病理性痛的镇痛作用以及天麻素对背根神经节Nav1.6通道的表达调控作用。方法:将60只雄性SD大鼠随机分为空白对照组、糖尿病组和天麻素处理组(10 mg·kg^-1·d^-1)。通过高脂饮食喂养4周,低剂量腹腔注射STZ(30 mg·kg^-1)的方法构建Ⅱ型糖尿病神经病理性痛大鼠模型,利用痛行为学检测观察各组大鼠的机械刺激足缩反应阈值变化,采用免疫荧光组织化学及Western blot方法观察各组大鼠背根神经节上Nav1.6通道的表达变化。结果:与空白对照组相比,糖尿病模型大鼠出现显著的机械刺激疼痛阈值下降(P<0.05),且模型组大鼠背根神经节神经元上的Nav1.6通道表达上调(P<0.05)。与糖尿病组相比,连续腹腔注射天麻素3天、7天、14天后,模型动物的疼痛明显缓解(P<0.05),另外天麻素可以翻转背根神经节上Nav1.6通道的高表达(P<0.05)。结论:天麻素可能通过降低Nav1.6通道的表达来缓解Ⅱ型糖尿病神经病理性疼痛,从而为天麻素缓解糖尿病神经病理性疼痛提供新的理论依据。     

Brain Indoleamines in Alloxan- and Streptozotocin-Induced Diabetic Rats

Previous work by other authors has shown that alloxan-induced diabetes increases whereas streptozotocin-induced diabetes does not alter nonesterified fatty acid (NEFA) plasma levels. The present study replicates these results and demonstrates that fasted, streptozotocin-induced diabetic animals also have increased NEFA levels. In addition, brain levels of 5-hydroxytryptamine (5-HT) and of its immediate precursor and metabolite were measured. Alloxan- and fasted, streptozotocin-induced diabetic rats showed significant increases in brain indoleamine concentrations, whereas fed, streptozotocin-induced diabetic rats had unchanged levels of the same compounds. Levels of brain indoleamines exhibited a strong positive correlation with wet-dog shakes (an index of 5-HT activity) elicited by hippocampal stimulation. Blockade of wet-dog shakes by 5-HT receptor antagonists strengthens the proposal that this behavior is a good index of central 5-HT activity. The increased content of brain indoleamines in alloxan- and fasted, streptozotocin-induced diabetic rats may be related to the increased NEFA plasma levels seen in the same animals. This hypothesis is supported by the positive correlation demonstrated between NEFA and 5-HT levels. In conclusion, it is suggested that alloxan-induced diabetes may represent a useful model for studying the various behavioral changes known to occur in diabetics.     

Destinations of Some Fast-Transported Proteins in Sensory Neurons of Bullfrog Sciatic Nerve

Many characteristics of proteins that are fast axonally transported have been described, but the destinations of most within the neuron remain unknown. We have studied the destinations of some fast-transported proteins in sensory neurons of the bullfrog sciatic nerve, specifically to determine which may be deposited in axons and which may be destined for more distal, possibly terminal, areas. Dorsal root ganglia were pulse-labeled with [35S]methionine in vitro, following which they were separated from the sciatic nerve. After additional periods of transport, radioactive proteins from two areas of the nerve were separated by two-dimensional polyacrylamide gel electrophoresis and used to develop x-ray film. The first area contained the wavefront of transported radioactivity (wavefront region), whereas the second area was taken from nerve through which the wavefront had already passed (plateau region). The amount of radioactivity in certain fast-transported protein species from each area was determined by computer analysis of digitized video images of fluorographs. Certain proteins were preferentially left behind the wavefront and, therefore, may supply axon and possibly other nerve components, whereas other proteins were found almost exclusively in the wavefront and, hence, may supply more distal, possibly terminal, areas.     

神经肽对体外培养人胚神经组织细胞生长发育的影响

本文成功地体外培养了人胚背根神经节和大脑皮层神经细胞,并首次通过它们研究了神经肽对种经组织细胞生长发育的影响。结果看出,培养液中加入神经肽后,背根神经节突起长度和密度增加;大脑皮层神经细胞存活数增加,分化神经元增多,胞体增大,突起增长,细胞生长加快,衰老减慢。超微结构观察表明,种经肽可增加细胞内的合成代谢和细胞间的相互连接,减少细胞变性损伤．本实验证实神经肽对神经组织具有种经营养作用,这为神经肽在临床上推广应用提供了实验依据。     

Intracerebroventricular Injection of Alarin Increased Glucose Uptake in Skeletal Muscle of Diabetic Rats

In order to investigate the central effect of alarin on glucose uptake, we administered alarin and/ or its inhibitor, ala6-25Cys into the cerebral ventricles of the type 2 diabetic rats. Then the relative parameters about glucose uptake in skeletal muscles were measured. We found that central treatment with alarin significantly increased the food intake, body weight and glucose infusion rates in hyperinsulinemic euglycemic clamp tests of the animals. Besides, the treatment also enhanced 2-deoxy-[³H]-D-glucose uptake, vesicle-associated membrane protein 2 contents, glucose transporter 4 protein and mRNA expression, as well as pAkt^Thr308, pAkt^Ser473 and total Akt levels in muscle cells, but reduced plasma glucose and insulin levels of the rats. All of the alarin-inducing events may be antagonised by central injection of ala6-25Cys. These results suggest that central administration of alarin stimulates glucose uptake mediated by activation of Akt signal pathway in type 2 diabetic animals.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.     

Expression and Activity of Cyclin-Dependent Kinase 5/p35 in Adult Rat Peripheral Nervous System

Abstract: In spite of the clarification in the temporal and spatial expression pattern of a cyclin-dependent kinase (Cdk) 5 and its neuron-specific activator, p35, in the CNS, it remains to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, we have examined their expression and activity in the PNS by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicated the expression of Cdk5 and p35 proteins in dorsal root ganglion (DRG) and sciatic nerve alike in the CNS. By immunohistochemistry, both proteins were shown to be present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicated the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity was found only in DRG, but not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains.     

Effects of Insulin on Altered Mechanical and Electrical Papillary Muscle Activities of Diabetic Rats

Since insulin compounds can restore some metabolic parameters and lipid profile alterations of the diabetic rat heart, we investigated whether these beneficial effects extend to diabetic rat cardiac dysfunctions. Twenty-four male Wistar albino rats, 6 months of age with an average body weight of 250–320 g, were divided randomly into three groups, each consisting of eight rats: control-group (C) rats were fed with standard rat nutrient and water; diabetic-group (D) rats were treated with a single intramuscular injection of streptozotocin (STZ, 45 mg/kg), dissolved in 0.01 M sodium citrate, pH adjusted to 4.5; and insulin-treated diabetic group (D + INS) rats were treated with subcutaneous injections of 1 IU/l insulin (INS) twice a day after a single intramuscular injection of STZ (45 mg/kg). Treatment of D rats with INS caused a time-dependent decrease in blood glucose. We found that the lipid profile and HbA_1c levels in the D + INS group reached the values of control rats at the end of the treatment period. Contraction force in group D was compared with values from groups C and D + INS (p < 0.05). Values were obtained at a muscle contraction and relaxation time of milliseconds, with contraction time in D compared to C and D compared to D + INS and C (p < 0.05). Rate-dependent changes in action potential configuration in left ventricular papillary muscle obtained from 8-week control, STZ-treated D and D + INS rats showed significant membrane potential changes between C and STZ-treated D animals. Action potential amplitude showed significant changes between matched D + INS and STZ-treated D animals. Depolarization time showed significant changes between C and STZ-treated D animals and between the D + INS and D groups. Half-repolarization time showed significant changes between D + INS and STZ-treated D animals and compared to the D and C groups. Our data suggest that the beneficial effects of insulin treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to restoration of the diminished K⁺ currents, partially related to the restoration of hyperglycemia.     

The Effect of Nimodipine on Calcium Homeostasis and Pain Sensitivity in Diabetic Rats

Summary 1. The pathogenesis of diabetic neuropathy is a complex phenomenon, the mechanisms of which are not fully understood. Our previous studies have shown that the intracellular calcium signaling is impaired in primary and secondary nociceptive neurons in rats with streptozotocin (STZ)-induced diabetes. Here, we investigated the effect of prolonged treatment with the L-type calcium channel blocker nimodipine on diabetes-induced changes in neuronal calcium signaling and pain sensitivity.2. Diabetes was induced in young rats (21 p.d.) by a streptozotocin injection. After 3 weeks of diabetes development, the rats were treated with nimodipine for another 3 weeks. The effect of nimodipine treatment on calcium homeostasis in nociceptive dorsal root ganglion neurons (DRG) and substantia gelatinosa (SG) neurons of the spinal cord slices was examined with fluorescent imaging technique.3. Nimodipine treatment was not able to normalize elevated resting intracellular calcium ([Ca²⁺] _i ) levels in small DRG neurons. However, it was able to restore impaired Ca²⁺ release from the ER, induced by either activation of ryanodine receptors or by receptor-independent mechanism in both DRG and SG neurons.4. The beneficiary effects of nimodipine treatment on [Ca²⁺] _i signaling were paralleled with the reversal of diabetes-induced thermal hypoalgesia and normalization of the acute phase of the response to formalin injection. Nimodipine treatment was also able to shorten the duration of the tonic phase of formalin response to the control values.5. To separate vasodilating effect of nimodipine Biessels et al., (Brain Res. 1035:86–93) from its effect on neuronal Ca²⁺ channels, a group of STZ-diabetic rats was treated with vasodilator – enalapril. Enalapril treatment also have some beneficial effect on normalizing Ca²⁺ release from the ER, however, it was far less explicit than the normalizing effect of nimodipine. Effect of enalapril treatment on nociceptive behavioral responses was also much less pronounced. It partially reversed diabetes-induced thermal hypoalgesia, but did not change the characteristics of the response to formalin injection.6. The results of this study suggest that chronic nimodipine treatment may be effective in restoring diabetes-impaired neuronal calcium homeostasis as well as reduction of diabetes-induced thermal hypoalgesia and noxious stimuli responses. The nimodipine effect is mediated through a direct neuronal action combined with some vascular mechanism.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.