Increase in fiber density for immunoreactive serotonin, substance P, enkephalin and thyrotropin-releasing hormone occurs during the early presymptomatic period of motoneuron disease in Wobbler mouse spinal cord ventral horn

The Wobbler mouse is a useful small animal model for the study of human motoneuron diseases. Besides showing the loss of motoneurons when the symptoms are expressed around the age of 3 weeks, we have also demonstrated the presumed ‘sprouting' of neuronal processes in the cervical spinal ventral horn which contain immunoreactive (IR) serotonin (5-HT), substance P (SP) and methionine and leucine enkephalins (ME, LE), as well as thyrotropin-releasing hormone (TRH). This occurs during the symptomatic period when IR-5-HT, ME and LE sprout at Stage 1, around the age of 3 weeks, whereas IR-SP sprouts only at a late stage (stage 4) of the disease (at age 3 months). The present investigation shows that the presumed sprouting occurs even before the appearance of symptoms and prior to significant motoneuron losses. IR-5-HT containing neuronal processes sprout by postnatal day 7, whereas IR-SP, -ME, -LE, and -TRH processes sprout by day 14. Hypothetically the early sprouts may contribute to the loss of motoneurons. They also respond to ciliary and brain derives neurotrophic factors cotreatment. IR-SP neuronal processes, although they sprout by day 14, show normal fiber density by the time symptoms appear (stage 1, age 21 days). However the SP sprouting is biphasic and a significant increase in number also occurs at an advanced stage of the disease (stage 4, age 3 months).     

Ultrastructural changes in blood vessels in epidermal growth factor treated experimental cutaneous wound model

This study investigates the impact of epidermal growth factor (EGF) on blood vessels, specifically on the development of intussusceptive angiogenesis in cutaneous wound healing. Excisional wounds were formed on both sides of the medulla spinalis in dorsal location of the rats. The control and EGF-treated groups were divided into two groups with respect to sacrifice day: 5 d and 7 d. EGF was topically applied to the EGF-treated group once a day. The wound tissue was removed from rats, embedded in araldite and paraffin, and then examined under transmission electron and light microscopes. The ultrastructural signs of intussusceptive angiogenesis, such as intraluminal protrusion of endothelial cells and formation of the contact zone of opposite endothelial cells, were observed in the wound. Our statistical analyses, based on light microscopy observations, also confirm that EGF treatment induces intussusceptive angiogenesis. Moreover, we found that induction of EGF impact on intussusceptive angiogenesis is higher on the 7th day of treatment than on the 5th day. This implies that the duration of EGF treatment is important. This research clarifies the effects of EGF on the vessels and proves that EGF induces intussusceptive angiogenesis, being a newer model with respect to sprouting type.     

Distribution of alpha and beta integrin subunits in the adult rat hippocampus after pilocarpine-induced neuronal cell loss,axonal reorganization and reactive astrogliosis

Integrins are –heterodimers that act as cell-extracellular matrix (ECM) and cell-cell adhesion molecules. During development, they are involved in axonal guidance, synaptogenesis, and in astrocytic maturation and migration. Here, we have examined the potential role of the integrin subunits 1–5 and 1–5 in axonal sprouting, synaptogenesis and reactive astrogliosis in the adult rat brain caused by pilocarpine-induced status epilepticus (SE). Strong hippocampal immunoreactivity of 1–5, 1, 3, 4, and 5 was observed in the pia mater, in vascular endothelia, and in astrocytes at the pial surface. 2 immunoreactivity was found exclusively in vascular endothelia. Pyramidal cells and interneurons of CA3–CA1, as well as hilar neurons revealed moderate 5 labeling in their cell bodies. Mossy fibers were immunoreactive for 2, 4, and 5. After pilocarpine-induced SE, strong immunoreactivity for 1, 2, 4, 5, 1, 3, and 4 was observed in reactive astrocytes. Our results show that members of the integrin family are differently distributed in cellular and subcellular compartments of the hippocampus and undergo specific patterns of regulation, which may be important for lesion-induced reactive changes in the adult brain.     

Evidence against cholera toxin B subunit as a reliable tracer for sprouting of primary afferents following peripheral nerve injury

In order to investigate whether cholera toxin B subunit (CTb) is transported by unmyelinated primary afferents following nerve injury, we transected the sciatic nerves of six rats, and injected the transected nerves (and in three cases also the intact contralateral nerves) with CTb, 2 weeks later. The relationship between CTb and two neuropeptides, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), was then examined in neurons in the ipsilateral L4 and L5 dorsal root ganglia, using immunofluorescence staining and confocal microscopy. We also immunostained sections of spinal cord and caudal medulla for CTb, NPY and VIP. Following nerve section, VIP immunoreactivity was increased in laminae I-II of the spinal cord while NPY immunoreactivity was increased in laminae III-IV of the spinal cord and in the gracile nucleus. On the contralateral side, CTb labelling was detected in laminae I and III-V of the dorsal horn of the L4 and L5 spinal segments, as well as in the gracile nucleus. CTb labelling was seen in the same areas on the lesioned side, but with a dramatic increase in lamina II. No VIP or NPY immunoreactivity was observed in L4 and L5 dorsal root ganglia on the side of the intact nerve, but on the lesioned side VIP was detected in many small neurons and NPY in numerous large neurons. In agreement with the report by Tong et al. [J. Comp. Neurol. 404 (1999) 143], we found that while CTb labelling in the dorsal root ganglion on the side of the intact nerve was mainly in large neurons, on the lesioned side CTb was present in dorsal root ganglion neurons of all sizes. The main finding of the present study was that almost all of the VIP- (96%) and NPY- (98%) positive neurons in the dorsal root ganglia on the lesioned side were also CTb-labelled. After nerve injury VIP is upregulated in fine afferents that terminate in laminae I and II, and most of these probably have unmyelinated axons. Since the cell bodies of these neurons were labelled with CTb that had been injected into the transected sciatic nerve, this suggests that many of these fine afferents, which do not normally transport CTb, are capable of doing so after injury.     

Sprouting of axon-like processes from axotomized retinal ganglion cells induced by normal and preinjured intravitreal optic nerve grafts

The failure of axonal regeneration in the mammalian central nervous system (CNS) is currently attributed to the glial environment of the lesion site which elaborates a multitude of inhibitory factors. Less attention has been paid to the potential of trophic support associated with the CNS, especially in relation to the status of the damaged CNS after an injury has been evoked. Using a grafting paradigm to implant an optic nerve (ON) segment into the vitreous, we have addressed how a prior damage of the ON before grafting influences its ability to stimulate retinal ganglion cells (RGCs) to sprout axon-like processes. Our results showed that a normal noninjured ON implanted intravitreally stimulated sprouting of RGCs, as revealed by sliver staining of the sprouting cells, as well as increasing the number of RGCs which express GAP-43. A prior crush injury of the ON 7 days before its implantation into the vitreous resulted in a significant decrease in its ability to stimulate RGC sprouting when the crush lesion segment was used as the graft, whereas grafts taken from segments proximal and distal to the lesion segment had potencies similar to that of the noninjured graft. Both astrocytes and oligodendrocytes were drastically reduced in number in the lesion segment graft, suggesting their involvement in the secretion of soluble trophic factors that may play a role in the sprouting and regeneration of damaged neurons.     

徐长卿种子质量的比较研究

目的考查不同生长状况下徐长卿种子的质量。方法测定种子的千粒重及发芽率。结果山东徐长卿种子的千粒重在3.63~6.17g,发芽率在85%~98%。26℃的温度易于种子萌发。结论2年生植株产的种子质量优于1年生植株的种子。随着时间的延长,徐长卿种子的发芽率受到一定影响。     

Epilepsy following cortical injury: Cellular and molecular mechanisms as targets for potential prophylaxis

The sequelae of traumatic brain injury, including posttraumatic epilepsy, represent a major societal problem. Significant resources are required to develop a better understanding of the underlying pathophysiologic mechanisms as targets for potential prophylactic therapies. Posttraumatic epilepsy undoubtedly involves numerous pathogenic factors that develop more or less in parallel. We have highlighted two potential "prime movers": disinhibition and development of new functional excitatory connectivity, which occur in a number of animal models and some forms of epilepsy in humans. Previous experiments have shown that tetrodotoxin (TTX) applied to injured cortex during a critical period early after lesion placement can prevent epileptogenesis in the partial cortical ("undercut") model of posttraumatic epilepsy. Here we show that such treatment markedly attenuates histologic indices of axonal and terminal sprouting and presumably associated aberrant excitatory connectivity. A second finding in the undercut model is a decrease in spontaneous inhibitory events. Current experiments show that this is accompanied by regressive alterations in fast-spiking γ-aminobutyric acid (GABA)ergic interneurons, including shrinkage of dendrites, marked decreases in axonal length, structural changes in inhibitory boutons, and loss of inhibitory synapses on pyramidal cells. Other data support the hypothesis that these anatomic abnormalities may result from loss of trophic support normally provided to interneurons by brain-derived neurotrophic factor (BDNF).
Approaches that prevent these two pathophysiologic mechanisms may offer avenues for prophylaxis for posttraumatic epilepsy. However, major issues such as the role of these processes in functional recovery from injury and the timing of the critical period(s) for application of potential therapies in humans need to be resolved.     

Alterations in noradrenergic innervation of the brain following dorsal bundle lesions in neonatal rats

Several seemingly conflicting sets of data have been reported on the regenerative capacity of central noradrenergic neurons, following transection of the ascending noradrenergic fiber tract in neonatal rats (Iacovitti et al., Dev Brain Res 1: 21-33, 1981; Jonsson and Sachs, Brain Res Bull 9: 641-650, 1982). In order to more fully investigate changes in noradrenergic neurons in the brain after such a transection, rats were lesioned at various times after birth, sometimes in conjunction with administration of the neurotoxin, 6-hydroxydopa (6-OHDOPA). Animals were sacrificed at 7, 10, 14, 28, 42 or 56 days after birth, in order to assess the pattern of noradrenergic neuronal damage, as well as the recovery rate. Dorsal bundle lesions were associated with neocortical and hippocampal hypoinnervation by noradrenergic fibers, and sprouting of a collateral fiber group, with production of noradrenergic hyperinnervation of the cerebellum and pons-medulla. Recovery of the norepinephrine (NE) content to control levels occurred in the neocortex at 8 weeks, when the dorsal bundle was lesioned at birth. When the lesion was produced at a later time (3 days or 5 days after birth), less recovery in the neocortex and hippocampus was found. Histofluorescent fiber number, as observed with a glyoxylic acid method, correlated with NE changes. It appears that 6-OHDOPA (20 micrograms/g IP) does not modify long-term recovery from a dorsal bundle lesion, when rats are co-treated at 3 days after birth. However, the length of the proximal noradrenergic fiber stump may be an important factor affecting the capacity for recovery from injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotrophic activity of proNGF in vivo

Ectopic expression of nerve growth factor (NGF) in transgenic mice results in the directional growth of sympathetic and/or sensory fibers. For instance, mice that over-express NGF under the control of the glial fibrillary acidic protein (GFAP) promoter exhibit robust axonal sprouting into the cerebellum, with no apparent loss of neurons in peripheral ganglia. Given the disagreement in the literature over whether pro-NGF exerts neurotrophic or apoptotic effects, we assessed the relative levels of proNGF and mature NGF in the cerebella of these transgenic mice. Blinded western blot analyses revealed that proNGF was the major species in both transgenic and wild type mice, with very low levels of mature NGF expression. While transgenic mice displayed significantly higher levels of cerebellar proNGF protein as compared to wild type mice, both strains possessed comparable levels of mature NGF. These data reveal that the ectopic expression of NGF in the cerebellum results in an increase in proNGF rather than mature NGF levels. Together with the robust axonal growth and lack of neuronal death in the ganglia in these animals, our results are clearly consistent with proNGF exhibiting neurotrophic activity in vivo.     

Physiologic and Morphologic Characteristics of Granule Cell Circuitry in Human Epileptic Hippocampus

Summary: Morphological and electrophysiological techiques were used to examine granule cells and their mossy fiber axons in nine surgically resected hippocampal specimens from temporal lobe epilepsy (TLE) patients. Timm histochemistry showed mossy fiber sprouting into the inner molecular layer (IML) of the dentate in a subset of tissue samples. In slices from five tissue samples, stimulus-induced bursting activity could be induced with a low concentration (2.5 μM) of bicuculline; bursts were sensitive to the N -methyl- d -aspartate (NMDA) blocker, APV. There was a general correlation between such sprouting and experimentally induced yperexcit ability. Fourteen granule cells from five tissue samples were intracellularly stained [with lucifer yellow (LY) or neurobiotin]. Axons from a subset of these neurons showed axon collaterals reaching into the IML, but this axon projection pattern for single cells was not directly correlated with degree of mossy fiber sprouting shown grossly by Timm staining. Electron microscopic examination of intracellularly stained elements showed mossy fiber axon terminals making asymmetric synaptic contacts (including autapses on the granule cell dendrite) with dendritic shafts and spines in both apical and basal domains. These data are consistent with the hypothesis that mossy fiber sprouting provides a structural basis for recurrent excitation of granule cells, but does not provide direct support of the hypothesis that mossy fiber sprouting causes hyperexcitability. The data suggest that granule cell bursting activity is at least in part a function of compromised synaptic inhibition, since levels of γ-aminobutyric acid (GABA) blockade that are generally subthreshold for burst induction were epileptogenic in some tissue samples from human epileptic hippocampus.