改良大鼠视网膜节细胞逆行标记方法的建立

目的　建立一种易于规范的、简易准确的视网膜节细胞 (retinalganglioncell,RGC)计数方法。方法　根据大鼠脑立体定位图谱确定双侧上丘和外侧膝状体在颅骨表面的投影 ,在投影处钻 4或 8个骨孔 ,用血管钳夹住微量进样器的尖端控制注射深度 ,注入 10g·L-1荧光金。 2周后取视网膜铺片在荧光显微镜下计数视网膜后极部、赤道部、远周部及背侧、尾侧、腹侧、嘴侧各象限RGC的数目 ,同时将视网膜铺片行甲苯酚紫染色后RGC计数。结果　所有动物均开颅手术标记成功。 4点、8点标记法标记的正常RGC的数目分别为 (2 2 19± 2 82 )个·mm-2 和 (2 2 0 7± 2 6 7)个·mm-2 ,2者无显著差异 ;四点法标记的大鼠RGC后极部、赤道部、远周部分别为 (2 4 99± 36 7)个·mm-2 、(2 2 15± 2 87)个·mm-2 、(194 4± 2 10 )个·mm-2 ,八点法标记的大鼠RGC后极部、赤道部、远周部分别为 (2 5 2 1± 35 7)个·mm-2 、(2 181±2 86 )个·mm-2 、(192 0± 190 )个·mm-2 ,其中后极部与远周部有显著差异 ;四点法标记的大鼠RGC背、尾、腹、嘴侧各象限RGC的数目分别为 (2 2 88± 30 3)个·mm-2 、(2 189± 2 77)个·mm-2 、(2 2 4 2± 2 6 7)个·mm-2 、(2 15 9± 30 5 )个·mm-2 ,八点法标记的大鼠RGC背、尾、腹、嘴侧各象限RGC的数目     

Alpha 7 nicotinic receptor subunit is present on serotonin neurons projecting to hippocampus and septum

The serotonergic transmitter system regulates hippocampal activity through its raphe projection to hippocampus and medial septum/diagonal band of Broca complex (MS/DBB), and most likely also indirectly through its interaction with the cholinergic neurotransmitter system. Nicotine, e.g., enhances hippocampal serotonin release probably through presynaptic nicotinic receptors. We investigated the possible presence of the alpha 7-nicotinic subunit on serotonergic neurons projecting to hippocampus and MS/DBB. By retrograde neuronal tracing, hippocampal serotonergic neurons were identified and with double fluorescence immunostaining and Alexa-488 bound alpha-bungarotoxin the presence of active alpha 7 receptor on their soma was determined. Most of the retrogradely labeled serotonin neurons contained the alpha 7 subunit. A low degree of colocalization between alpha-bungarotoxin and serotonin-positive neurons suggest that the alpha 7 subunit may be transported anterogradely to the serotonergic axonal terminals.     

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.     

Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve

Even though peripheral nerves regenerate well, axons are often misrouted and reinnervate inappropriate distal pathways post-injury. Misrouting most likely occurs at branch points where regenerating axons make choices. Here, we show that the accuracy of sensory axon reinnervation is enhanced by overexpression of the guidance molecule nerve growth factor (NGF) distal to the bifurcation. We used the femoral nerve as a model, which contains both sensory and motor axons that intermingle in the parent trunk and distally segregate into the saphenous (SB) and motor branches (MB). Transection of the parent trunk resulted in misrouting of axon reinnervation to SB and MB. To enhance sensory axon targeting, recombinant adenovirus encoding NGF was injected along the SB close to the bifurcation 1 week post-injury. The accuracy of axon reinnervation was assessed by retrograde tracing at 3 or 8 weeks after nerve injury. NGF overexpression significantly increased the accuracy of SB axon reinnervation to the appropriate nerve branch, in a manner independent of enhancing axon regeneration. This novel finding provides in vivo evidence that gradient expression of neurotrophin can be used to enhance targeting of distal peripheral pathways to increase axon regeneration into the appropriate nerve branch.     

Trigeminal transition zone/rostral ventromedial medulla connections and facilitation of orofacial hyperalgesia after masseter inflammation in rats

Recent studies have implicated a role for the trigeminal interpolaris/caudalis (Vi/Vc) transition zone in response to orofacial injury. Using combined neuronal tracing and Fos protein immunocytochemistry, we investigated functional connections between the Vi/Vc transition zone and rostral ventromedial medulla (RVM), a key structure in descending pain modulation. Rats were injected with a retrograde tracer, FluoroGold, into the RVM 7 days before injection of an inflammatory agent, complete Freund's adjuvant, into the masseter muscle and perfused at 2 hours postinflammation. A population of neurons in the ventral Vi/Vc overlapping with caudal ventrolateral medulla, and lamina V of the trigeminal subnucleus caudalis (Vc), exhibited FluoroGold/Fos double staining, suggesting the activation of the trigeminal-RVM pathway after inflammation. No double-labeled neurons were found in the dorsal Vi/Vc and laminae I-IV of Vc. Injection of an anterograde tracer, Phaseolus vulgaris leucoagglutinin, into the RVM resulted in labeling profiles overlapped with the region that showed FluoroGold/Fos double labeling, suggesting reciprocal connections between RVM and Vi/Vc. Lesions of Vc with a soma-selective neurotoxin, ibotenic acid, significantly reduced inflammation-induced Fos expression as well as the number of FluoroGold/Fos double-labeled neurons in the ventral Vi/Vc (P<0.05). Compared with control rats, lesions of the RVM (n=6) or Vi/Vc (n=6) with ibotenic acid led to the elimination or attenuation of masseter hyperalgesia/allodynia developed after masseter inflammation (P<0.05-0.01). The present study demonstrates reciprocal connections between the ventral Vi/Vc transition zone and RVM. The Vi/Vc-RVM pathway is activated after orofacial deep tissue injury and plays a critical role in facilitating orofacial hyperalgesia.     

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.     

大鼠视神经部分损伤后自发再生的实验研究

目的观察大鼠视神经部分损伤后自发再生纤维的形成。方法采用荧光金顺行标记和生长相关蛋白免疫电镜的方法观察反向夹持镊损伤视神经后的再生纤维情况。结果视神经损伤2周后，损伤点后0．5mm内有较密集的荧光金标记，损伤远端散在条状、线状荧光；损伤区前的神经纤维的朗飞氏结的轴突内及损伤区后的无髓神经纤维内有生长相关蛋白-43的阳性表达。生长相关蛋白-43位于轴浆内呈区域性或，临近轴膜分布。结论大鼠视神经部分损伤后具有自发再生能力。     

GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap

The present work was performed to determine the ability of neurotrophic factors to allow axonal regeneration across a 15-mm-long gap in the rat sciatic nerve. Synthetic nerve guidance channels slowly releasing NGF and GDNF were fabricated and sutured to the cut ends of the nerve to bridge the gap. After 7 weeks, nerve cables had formed in nine out of ten channels in both the NGF and GDNF groups, while no neuronal cables were present in the control group. The average number of myelinated axons at the midpoint of the regenerated nerves was significantly greater in the presence of GDNF than NGF (4942 +/-1627 vs. 1199 +/-431, P < or = 0.04). A significantly greater number of neuronal cells in the GDNF group, when compared to the NGF group, retrogradely transported FluoroGold injected distal to the injury site before explantation. The total number of labelled motoneurons observed in the ventral horn of the spinal cord was 98.1 +/-23.4 vs. 20.0 +/-8.5 (P < or = 0.001) in the presence of GDNF and NGF, respectively. In the dorsal root ganglia, 22.7% +/- 4.9% vs. 3.2% +/-1.9% (P +/-0.005) of sensory neurons were labelled retrogradely in the GDNF and NGF treatment groups, respectively. The present study demonstrates that, sustained delivery of GDNF and NGF to the injury site, by synthetic nerve guidance channels, allows regeneration of both sensory and motor axons over long gaps; GDNF leads to better overall regeneration in the sciatic nerve.     

Streptozotocin-induced diabetes reduces retrograde axonal transport in the afferent and efferent vagus nerve

Diabetes-induced alterations in nerve function include reductions in the retrograde axonal transport of neurotrophins. A decreased axonal accumulation of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of streptozotocin (STZ)-induced diabetic rats was previously shown. In the current study, no changes in the NGF and NT-3 protein or mRNA levels in the stomach or atrium, two vagally innervated organs, were noted after 16 or 24 weeks of diabetes. Moreover, the amounts of neurotrophin receptor (p75, TrkA, TrkC) mRNAs in the vagus nerve and vagal afferent nodose ganglion were not reduced in diabetic rats. These data suggest that neither diminished access to target-derived neurotrophins nor the loss of relevant neurotrophin receptors accounts for the diabetes-induced alteration in the retrograde axonal transport of neurotrophins. To assess whether diabetes causes a defect in axonal transport that may not be specific to neurotrophin transport, we studied the ability of a neuronal tracer (FluoroGold, FG) to be retrogradely transported by vagal neurons of control and diabetic rats. After vagal target tissue (stomach) injections of FG, the numbers of FG-labeled afferent and efferent vagal neurons were counted in the nodose ganglion and in the dorsal motor nucleus of the vagus, respectively. After 24 weeks of diabetes, FG was retrogradely transported to more than 50% fewer afferent and efferent vagal neurons in the STZ-diabetic compared to control rats. The diabetes-induced deficit in retrograde axonal transport of FG is likely to reflect alterations in basic axonal transport mechanisms in both the afferent and efferent vagus nerve that contribute to the previously observed reductions in neurotrophin transport.     

Neural reflex pathway between cervical spinal and sympathetic ganglia in rabbits: implication for pathogenesis of cervical vertigo

Background contextA functional association between cervix and vertigo has been observed in patients with cervical vertigo, implicating correlation between cervical spinal and sympathetic ganglia. However, it is unclear where there is an anatomic connection between those two groups of ganglia.PurposeThis study aimed to investigate the existence of the neural connections between cervical spinal and sympathetic ganglia.Study design/settingFluoroGold staining patterns in cervical spinal and sympathetic ganglia were evaluated using FluoroGold retrograde tracing in New Zealand rabbits.MethodsNew Zealand rabbits were randomly divided into superior cervical spinal ganglion injection groups, inferior cervical spinal ganglion injection groups, superior cervical sympathetic ganglion injection group, and inferior cervical sympathetic ganglion injection group. Four percent FluoroGold solution was injected into these ganglia. Distribution of FluoroGold in cervical spinal and sympathetic ganglia was observed under a microscope.ResultsWhen FluoroGold solution was injected into C2 and C3 superior cervical spinal ganglia or C5–C6 inferior cervical spinal ganglia, fluorescence was only observed in the ipsilateral superior or inferior cervical sympathetic ganglia, respectively. When FluoroGold solution was injected into superior or inferior cervical sympathetic ganglia, fluorescence was found mainly in the ipsilateral C3–C4 superior or C5–C8 inferior spinal ganglia. No fluorescence was observed in contralateral ganglia of experimental animals and all ganglia of matched control animals injected with physiological saline.ConclusionsBidirectional nerve fiber connections between cervical spinal and sympathetic ganglia were observed, and these connections are arranged in a segmental distribution. This observation may provide a possible neuroanatomic basis for the pathogenesis of cervical vertigo.