Correlation between CGRP immunoreactivity and firing activity in cat abducens motoneurons

Intercellular communication through gap junction channels is a prominent feature of the developing cerebral cortex. In the first 2 weeks after birth, a time critical in the development of the rat neocortex, extensive cell coupling has been documented that diminishes as the cortex matures. Among the family of gap junction proteins, connexins 26, 36, and 43 are differentially expressed during cortical development. We used intracellular dye injections and connexin immunohistochemistry to investigate the coupling patterns and connexin expression between the different neuronal and glial cell types of the developing cortex of the rat. We found that neurons and glia couple homotypically and heterotypically at postnatal days 7 and 14. Although the prevalence of coupling was homotypic, there was considerable heterotypic coupling that involved pyramidal and nonpyramidal neurons, the principal neuronal cell types of the cortex, or neurons and astrocytes. Coupling between different cell types appeared to be mediated by differential expression of connexins 26, 36, and 43. It may be that coupling between cells in the developing neocortex is a function of the spatial and temporal expression of these and other connexin proteins.     

Recombinant C fragment of botulinum neurotoxin B serotype (rBoNTB (HC)) immune response and protection in the rhesus monkey

Botulinum neurotoxin B (BoNTB) is a distinct protein subtype of a family of neurotoxins with the potential for use in biological warfare or terrorist attacks. This study is one in a series evaluating the immunogenicity and protective effects of recombinant vaccines against the different subtypes of botulinum toxin. The recombinant subunit vaccines encoding the C fragment portion (50 kDa) of the toxins are produced in the yeast, Pichia pastoris. In this study, groups of rhesus monkeys were vaccinated with three doses (1 and 5 μg per dose) of rBoNTB(H_c) vaccine. Total and neutralizing antibody titers were determined at various times during and postvaccination. Two groups of vaccinated monkeys plus non-vaccinated controls were actively challenged with B toxin by aerosol exposure. All monkeys receiving vaccine were protected from the toxin and no clinical signs of disease were observed, while controls displaying classic signs of botulism succumbed to the toxin challenge. Two additional groups of monkeys receiving the same vaccine regiment as the first two groups had significant levels of circulating neutralizing antibody titers up to 24 months postvaccination. This non-human primate study demonstrated the short- and long-term immunity afforded by the rBoNTB(H_c) vaccine.     

Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P.

Episodes of prolonged seizures or head trauma produce chronic hippocampal network hyperexcitability hypothesized to result primarily from inhibitory interneuron loss or dysfunction. The possibly causal role of inhibitory neuron failure in the development of epileptiform pathophysiology remains unclear because global neurologic injuries produce such a multitude of effects. The recent finding that Substance P receptors (SPRs) are expressed exclusively in the rat hippocampus by inhibitory interneurons provided the rationale for attempting to ablate interneurons selectively by using neurotoxic conjugates of SPR ligands and the ribosome inactivating protein saporin that specifically target Substance P receptor-expressing cells. Whereas intrahippocampal microinjection of a conjugate of native SP and saporin produced significant nonspecific damage at concentrations needed to produce even limited selective loss of SPR-positive cells, a conjugate of saporin and the more potent and peptidase-resistant SP analog [Sar(9), Met(O(2))(11)] Substance P (SSP-saporin) caused negligible nonspecific damage at the injection site, and a virtually complete loss of SPR-like immunoreactivity (LI) up to 1 mm from the injection site. Within the SPR depletion zone, immunoreactivities for most GABA-, parvalbumin-, somatostatin-, and cholecystokinin-immunoreactive cells and fibers were eliminated. The few interneurons detectable within the affected zone were devoid of SPR-LI. The apparent loss of interneurons was selective in that calbindin- and glutamate receptor subunit 2 (GluR2) -positive principal cells survived within the affected zone, as did myelinated fibers and the extrinsic calretinin- and tyrosine hydroxylase--immunoreactive terminals of subcortical afferents. An apparent lack of reactive synaptic reorganization in response to interneuron loss was indicated by zinc transporter-3 (ZnT3)-- and beta-synuclein--LI, as well as by Timm staining, all of which revealed relatively normal patterns of excitatory terminal distribution. Control injections produced minor damage at the injection site, but no apparent specific loss of SPR-LI. One to 12 weeks after injection of SSP-saporin, extracellular electrophysiological field responses recorded in the CA1 pyramidal and dentate granule cell layers in response to afferent stimulation were blindly evaluated simultaneously in two sites 1-2 mm apart along the longitudinal hippocampal axis. SSP-saporin-treated rats exhibited relatively normal responses in some sites, whereas disinhibition and hyperexcitability indistinguishable from the pathophysiology produced by experimental status epilepticus were simultaneously recorded at adjacent sites. Anatomic analysis of the recording sites in each animal revealed that epileptiform pathophysiology was consistently observed only within areas of SPR ablation, whereas relatively normal evoked responses were recorded from immediately adjacent and relatively unaffected regions. These data establish the efficacy of [Sar(9), Met(O(2))(11)] Substance P-saporin for producing a selective and spatially extensive ablation of hippocampal inhibitory interneurons in vivo and a highly focal disinhibition that was restricted to the site of interneuron loss. These results also demonstrate that the "epileptic" pathophysiology produced by experimental status epilepticus or head trauma can be replicated by focal interneuron loss per se, without involving principal cell loss and other interpretive confounds inherent in the use of global neurologic injury models.     

眼镜蛇毒因子激活补体对大鼠外周血白细胞数目和功能的影响

目的：观察眼镜蛇毒因子（ＣＶＦ）激活补体对大鼠外周血白细胞数目和功能的影响。方法：采用吞噬金葡菌法、玻璃血球计数板法分析测定白细胞的吞噬和粘附功能。结果：ＣＶＦ１．０ｍｇ·ｋｇ－１ｉｐ，能使外周血白细胞吞噬和粘附功能显著升高，白细胞的吞噬率从２０．７％±４．９％上升至４２．５％±５．５％，粘附率从７３．４％±８．３％上升至９３．６％±９．８％；同时外周血白细胞数目短暂下降后显著增多。结论：ＣＶＦ激活补体可使外周血白细胞数明显改变并显著增强其功能，提示补体可能参与白细胞功能的调节。     

A comparison of the spread of three formulations of botulinum neurotoxin A as determined by effects on muscle function

The purpose of these experiments was to compare the spread of three formulations of botulinum neurotoxin A. A gelatin/phosphate buffer (C), Dysport^R (D {0.5 U}, Botox^R (B {0.167 U}), or a purified preparation of botulinum neurotoxin A [Bont A] (BA {0.5 U}) was injected into the tibialis anterior of male, Wistar rats. After 4 days, the adjacent extensor digitorum longus muscle was isolated in situ and the nerve was maximally stimulated to determine contractile properties and the rate of fatigue. There were no differences in body or muscle weights between any of the groups after 4 days of treatment. Maximal twitch and tetanic tensions were decreased ≈ 25% ( p < 0.05) in all treatment groups compared to C. In addition, rate of tension development was significantly less in all treatment groups compared to C but one-half relaxation time and time to peak tension were not different between any groups. Fatigue of the muscle was significantly faster in all groups compared to C but there was no difference between treatment groups. These data indicated that botulinum toxin A injected intramuscularly was likely to spread to adjacent muscles but that the spread was not different between the three formulations tested. The effect of the spread ranged from a slight to a severe reduction in maximal tension but this did not occur in all animals studied.     

Characterization of a Neurite Outgrowth Inhibitor Expressed After CNS Injury

Reactive gliosis, a general response to injury in the central nervous system grey and white matter, represents a serious obstacle to axonal regeneration in mammals. In culture, myelin-free plasma membranes from normal rat brain tissue promoted neurite outgrowth, whereas myelin-free membranes purified from injured tissue were inhibitory. The inhibitory activity could be solubilized by detergent, was sensible to glycosaminoglycan lyase digestion and eluted with an apparent molecular weight of 160 – 220 kDa in gel filtration chromatography. When presented as a surface-bound molecule, the inhibitor prevented neurite initiation; when added in a soluble form to growing neurites, it induced their retraction. These results provide cellular and molecular evidence supporting the classical view that, in the mammalian central nervous system, damage-evoked gliosis correlates with the expression of molecules capable of preventing neurite outgrowth.     

Recurrent paraplegia after remyelination of the spinal cord

We have conducted a long-term study of spinal cord morphology and motor function recovery in rats that have undergone lumbar spinal demyelination induced by the B-fragment of cholera toxin (CTB)-saporin. We found that, after the initial demyelination and paraplegia, motor function recovered and was stable for up to 9 months, after which there occurred a slow deterioration of motor function accompanied by loss of motoneurons and loss of spinal white matter. A striking morphological feature was the appearance of large spheroids of calcium in the ventral and dorsal horns and occasionally in the white matter. Motor performance deterioration occurred earlier and was more severe in rats that had been exercised on a treadmill, but the same morphological changes occurred in both exercise- and nonexercise-treated animals. Rats given treadmill exercise starting 3 weeks after toxin injection had a mean motor deficit score of 3.0 (i.e., paraplegia) at perfusion, whereas the nontreadmill-treated rats had a mean score of 1.8 (SD 0.38; n = 6; P <.05). These findings suggest that, in addition to the acute effects of the toxin-induced demyelination from which there is recovery of motor function, there are chronic irreversible effects of the toxin, or the initial demyelination, that cause a slow progressive degeneration of the spinal cord. This model might therefore be useful in studying the long-term effects of spinal insult of the type associated with conditions such as postpolio syndrome.     

Use of Biophysical Characterization in Preformulation Development of a Heavy-Chain Fragment of Botulinum Serotype B: Evaluation of Suitable Purification Process Conditions

PURPOSE: The purpose of this study was to investigate the physicochemical and structural characteristics of recombinant botulinum serotype B (rBoNTB(Hc)) under various conditions and to use the information in evaluating suitable purification process conditions. METHODS: The solubility of rBoNTB(Hc) was evaluated at pH 4, 5, 6 7.5, 8, and 9. Secondary structure was evaluated using circular dichroism, and conformational stability was monitored using highsensitivity differential scanning calorimetry. Hydrophobic interaction chromatography, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), peptide mapping, and UV spectroscopy were used to monitor stability under the various conditions. RESULTS: The secondary structure of rBoNTB(Hc) consists predominantly of beta-sheets. Solubility of rBoNTB(Hc) was lowest at its pI and highest at low and high pH. In the presence of NaCl, however, solubility decreased with increase in pH. Conformational and chemical stability are improved below pH 7.5. In the presence of 150 mM NaCl at high pH, conformational and chemical stability of rBoNTB(Hc) are further decreased. The study suggests that the purification process should minimize exposure of rBoNTB(Hc) to high pH and salt conditions. CONCLUSIONS: Optimal stability of rBoNTB(Hc) is achieved at low pH. The biophysical and analytical studies provide us with an understanding of rBoNTB(Hc) stability behavior in solution and assists in developing efficient purification conditions.     

中华眼镜蛇毒诱导HL-60细胞凋亡及其机制探讨

目的　观察中华眼镜蛇毒体外对白血病细胞凋亡的影响 ,并探讨其可能的作用机制。方法　本研究采用血清药理学方法 ,以人白血病细胞HL 60细胞为靶细胞 ,通过流式细胞仪分析、DNA片段凝胶电泳、免疫组织化学 (S P法 )观察中华眼镜蛇毒兔血清体外对细胞凋亡的诱导作用及癌基因表达的影响。结果　经DNA电泳、流式细胞仪分析显示 :中华眼镜蛇毒兔血清与HL 60细胞共同培养 48h可诱导其细胞凋亡增加 ,bcl 2、c myc基因蛋白表达下调。结论　中华眼镜蛇毒兔血清体外抑制HL 60细胞增殖可能与其诱导细胞凋亡及使细胞bcl 2、c myc基因蛋白表达下调密切相关