Jan Westman

Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells

Present techniques for the intracellular staining of physiologically identified neurons are not quite satisfactory for ultrastructural investigations. Iontophoretic administration of cobalt chloride for subsequent reaction with ammonium sulphide 12 does not allow fixation before incubation, a disadvantage as regards the ultrastructural preservation. If the injected cobalt chloride is allowed to react with diaminobenzidine 4 the fine structure of the cytoplasm appears to be damaged. This holds true also for iontophoretically injected Procion brown 2. Procion yellow 1~ is not electron dense and ultrastructural identification has to depend on changes of cell organelles9, la. Radioautographic techniques 5 are laborious because ultrastructural interpretation needs statistical analysis since the silver grains are situated at a distance from the source of activity 14. In this study the enzyme horseradish peroxidase (HRP) has been used for intracellular staining. HRP has recently found extensive use as a neuronal marker (for references, see ref. 10) in light and electron microscopical studies. The histochemical staining procedure employed seems not to damage the ultrastructure and the product of enzyme reaction is found in lysosome-like organelles 1°. In the present investigation the enzyme was administered intracellularly in spinocervical tract cells a, neurons previously studied with intracellular injections of Procion yellow 1,s. Cells were identified by monosynaptic excitation from the cutaneous nerves and by antidromic invasion from the dorsolateral funiculus in segment C3 (Fig. 1A-G). An additional criterion was the absence of antidromic invasion from C1 except at a high threshold. A total of 23 spinocervical tract cells were impaled with single barrelled micropipettes with tip diameters of 2-3/~m. The electrodes were filled by pressure with a 15-20 ~o (w/v) solution of HRP (Sigma, type II). In contrast to previous studies, where extracellular iontophoresis of HRP was performed 7,11, solutions in distilled water were initially used. In order to decrease the high electrode resistance by increasing ionic strength, both acid and basic solutions were tested. The best results were obtained

Pathophysiology of brain edema and cell changes following hyperthermic brain injury

Publisher Summary This chapter focuses on brain edema formation in heat stress and its contribution to cell injury. In addition, the basic concepts of brain edema formation, including various biomechanical and biophysical factors, and chemical mediators involved in this mechanism are reviewed. Based on the experimental observations, a new pharmacological approach to reduce edema formation and cell injury by modifying the breakdown of the blood–brain barrier in heat stress is discussed. Brain edema is a serious complication of various neurological diseases following traumatic, ischemic, or hypoxic injuries to the central nervous system. The early clinical symptoms of brain edema comprise signs of headache, nausea, vomiting, disturbances of consciousness, and occasionally coma. Progression of edema leads to brain herniation and vascular infarction that are common causes of death. The effects of hyperthermia on brain dysfunction is reduced by advancing age, anaesthesia, or prior heat experience indicating that the age and physiological states of the animals before heat exposure are important determining factors for the outcome of thermal brain damage.

Involvement of nitric oxide in acute spinal cord injury: an immunocytochemical study using light and electron microscopy in the rat

The possibility that nitric oxide participates in the pathophysiology of spinal cord injury was examined using a constitutive isoform of neuronal nitric oxide synthase immunoreactivity in a rat model. Spinal cord trauma was produced by making an incision into the right dorsal horn of the T10-11 segments. Five h after trauma, a marked upregulation of NOS-immunostained neurons was seen in the perifocal T9 and T12 segments of the cord. The immunolabelling was most pronounced in the dorsal horn of the ipsilateral side. Topical application of an antiserum to nitric oxide synthase (NOS) 2 min after injury prevented the trauma-induced upregulation of NOS-immunoreactivity. In contrast, application of preabsorbed serum or L-NAME, an inhibitor to NOS, was ineffective in reducing the induction of NOS-immunoreactivity. Trauma caused a marked expansion of the cord and resulted in marked cell changes. This expansion and cell reaction was significantly reduced following application of NOS antiserum but it was not seen after application of preabsorbed antiserum or L-NAME. Our results for the first time show that a focal trauma to the spinal cord has the capacity to upregulate neuronal NOS immunoreactivity and that application of NOS antiserum has a neuro protective effect. This indicates that nitric oxide is somehow involved in the pathogenesis of secondary injuries after spinal cord trauma.

Effects of glucose on the ultrastructure and insulin biosynthesis of isolated mouse pancreatic islets maintained in tissue culture

SummaryIsolated mouse pancreatic islets were maintained in tissue culture for up to 12 days in glucose concentrations varying between 3.3 and 28 ml. A satisfactory ultrastructural preservation of the islet cells was found irrespective of the glucose concentration of the culture medium. While the B-cells of islets cultured in the lower glucose concentration showed slight degranulation, there was extensive degranulation and increased amounts of rough-surfaced endoplasmic reticulum after culture in the higher glucose concentration. The immunoreactive insulin content of islets cultured at 28 mM glucose was markedly decreased and the insulin secretion during the culture period was much higher than that of islets cultured at 3.3 mM glucose. The insulin biosynthesis, as reflected in the incorporation of3H-leucine into gel chromatographed extracts of acid-ethanol soluble islet proteins, was studied either during the culture period or at the end of a 6-day culture in short-term incubations lasting for 90 or 180 min. The results consistently showed that the biosynthesis of insulin proceeded at a high rate and remained regulated by glucose throughout the culture period. The continuous addition of newly synthesized and labelled insulin to the small intracellular insulin pool of the high-glucose cultured B-cells produced a very high specific radioactivity of the insulin.

Brain derived neurotrophic factor and insulin like growth factor-1 attenuate upregulation of nitric oxide synthase and cell injury following trauma to the spinal cord

SummaryThe possibility that brain derived neurotrophic factor (BDNF) and insulin like growth factor-1 (IGF) induced neuroprotectivn is influenced by mechanisms involving nitric oxide was examined in a rat model of focal spinal cord injury. BDNF or IGF-I (0.1 μg/10 [1 in phosphate buffer saline) was applied topically 30 min before injury on the exposed spinal cord followed by repeated doses of growth factors immediately before and 30 min after injury. Thereafter application of BDNF or IGF was carried out at every 1 h interval until sacrifice. Five hours after injury, the tissue pieces from the T9 segment were processed for nNOS immunostaining, edema and cell injury. Untreated injured rats showed a profound upregulation of nNOS which was most pronounced in the nerve cells of the ipsilateral side. A marked increase in the blood-spinal cord barrier (BSCB) permeability to125I-albumin, water content and cell injury in these perifocal segments was also found. Pretreatment with BDNF and IGF significantly reduced the upregulation of nNOS in the spinal cord. This effect of the growth factors was most pronounced in the contralateral side. Rats treated with these neurotrophic factors showed much less signs of BSCB damage, edema and cell injury. These results suggest that BDNF and IGF pretreatment is neuroprotective in spinal cord injury and that these neurotrophic factors have the capacity to down regulate nNOS expression following trauma to the spinal cord. Our data provide new experimental evidences which suggest that BDNF and IGF may exert their potential neuroprotective effects probably via regulation of NOS activity.

Role of nitric oxide in blood-brain barrier permeability, brain edema and cell damage following hyperthermic brain injury. An experimental study using EGB-761 and Gingkolide B pretreatment in the rat.

The role of oxidative stress in hyperthermia induced upregulation of constitutive and inducible isoforms of nitric oxide synthase (NOS) in the central nervous system (CNS) was investigated using immunohistochemistry in a rat model. Exposure of rats to heat stress at 38 degrees C for 4 h resulted in marked upregulation of constitutive NOS (cNOS) and a mild but significant expression of inducible NOS (iNOS) in several brain regions exhibiting leakage of the blood-brain barrier (BBB), brain edema formation and cell injury. Pretreatment with the potent antioxidative compound EGB-761 or its constituent, Ginkgolide B significantly attenuated upregulation of cNOS and iNOS in the brain and also reduced the BBB permeability disturbances, brain edema and cell injury. These neuroprotective effects were most marked in the EGB-761 pretreated rats. Our observations strongly suggest that (i) EGB-761 and Ginkgolide B pretreatment offer significant neuroprotection in hyperthermic brain injury, (ii) upregulation of cNOS and iNOS are injurious to the cell and, (iii) oxidative stress plays an important role in NOS expression and cell injury.

The lateral cervical nucleus in the cat. IV. A light and electron microscopical study after midbrain lesions with demonstration of indirect wallerian degeneration at the ultra-structural level.

SummaryThe lateral cervical nucleus (LCN) has been studied with the light and electron microscope after midbrain lesions in kittens of different ages, and in adult cats. It has been shown that nerve cells remain within the contralateral LCN after hemisection of the midbrain. performed on 1 day old kittens. The estimated number of these remaining cells was 3–19% in the different cases. No fibers descending to the LCN from levels rostral to the midbrain could be demonstrated in adult cats with the Nauta technique. A combined Nauta and electron microscopical investigation was performed on kittens operated on at different ages and allowed to survive varying times postoperatively. At the operations the LCN-axons were transected at midbrain level. The Nauta investigation demonstrated silver impregnation of degenerating LCN-neurons on the affected side of the same type as has been described recently in other neuronal systems following axonal transection. The ultrastructural study revealed electron dense degenerating dendrites and probably also nerve cells within the LCN. The dense degenerative changes were very similar to the dense degeneration in terminal boutons following transection of parent axons. The potential value of the findings for electron microscopical research in neuroanatomy is discussed. The results in terms of connections of the LCN are discussed in the light of known anatomical and physiological data of the LCN.

The projection to the mesencephalon from the sensory trigeminal nuclei. An anatomical study in the cat

The terminal areas and the cells of origin of the projection from the sensory trigeminal nuclei to the mesencephalon were investigated, using the method of anterograde and retrograde transport of horseradish peroxidase or wheat germ agglutinin-horseradish peroxidase conjugate. Injection of tracer into the nucleus interpolaris or nucleus oralis (in the latter cases with involvement of the nucleus principalis) resulted in dense anterograde labeling in the deep and intermediate gray layers of the contralateral superior colliculus, extending throughout the rostrocaudal extent of the colliculus with the exception of its caudalmost part, which was not labeled. Minor projections to the intercollicular nucleus, posterior pretectal nucleus and nucleus of Darkschewitsch were found. Injection of tracer into the nucleus caudalis yielded a completely different result; terminal labeling in the midbrain was now present only in the periaqueductal gray matter, in its rostral and middle parts. The retrograde labeling observed after injection of tracer into the midbrain terminal areas showed that the cells of origin were located mainly in the alaminar spinal trigeminal nucleus, and the highest density of labeled neurons was found in the rostral part (subnucleus y) of the nucleus oralis. The retrograde labeling in the nucleus principalis was very sparse and almost exclusively involved peripherally located neurons. In the nucleus caudalis the overwhelming majority of the retrogradely labeled neurons were situated in its marginal layer. The functional implications of the above observations are discussed in relation to the findings in previous studies of the projections from the dorsal column nuclei and spinal cord to the midbrain. The combined results suggest that the trigeminal projections to the superior colliculus may be involved in the mechanisms of orientational behavior. The observation that the projection to the periaqueductal gray matter originates in the marginal layer suggests that it transmits information related to noxious stimuli.

Topical application of TNF-α antiserum attenuates spinal cord trauma induced edema formation, microvascular permeability disturbances and cell injury in the rat

The possibility that antiserum to tumour necrosis factor-alpha (TNF-alpha) is neuroprotective in spinal cord injury (SCI) was examined in a rat model. SCI was produced by making an incision into the right dorsal horn at the T10-11 segments. Top TNF-alpha antiserum at three concentrations (1:10; 1:50 and 1:100) given 30 min before or 2 min, 5 min or 10 min after trauma resulted in marked reduction in visible swelling, edema formation, and leakage of radiolabelled iodine tracer within the T9 and T12 segments at 5 h in a dose dependent manner. This neuroprotective effect was most pronounced when the antiserum at the highest dose level (1:10) was applied 10 min after SCI. The TNF-alpha antiserum also reduced the SCI induced upregulation of neuronal nitric oxide synthase (nNOS) immunoreactivity in a concentration dependent manner. Taken together, these results suggest that local application of TNF-alpha antiserum is neuroprotective in SCI and that this effect is mediated through NOS regulation.

Role of Serotonin and Prostaglandins in Brain Edema Induced by Heat Stress. An Experimental Study in the Young Rat

The possibility that serotonin and prostaglandins participate in edema formation following heat stress (HS) was examined in young rats. Exposure of conscious young animals (8-9 weeks old) to heat at 38 degrees C in a biological oxygen demand (BOD) incubator (relative humidity 50-55%; wind velocity 20-25 cm/s) for 4 h resulted in marked increase in the whole brain water content (about 3%) as compared to animals kept at room temperature (21 degrees C). A marked extravasation of Evans blue and 131I-sodium occurred in the brain of heat exposed animals as compared to normal animals. Morphological examination using electron microscopy of selected brain regions of heat stressed animals showed profound cell changes. Thus perivascular edema, swollen neuronal and glial cells, membrane damage, vesiculation of myelin, axonal swelling and synaptic damage was frequent in this group of untreated animals. Pretreatment with ketanserin (a selective serotonin2 receptor antagonist) or indomethacin (an inhibitor of prostaglandin synthesis) markedly reduced edema formation after 4 h HS in young animals. These heat stressed animals had considerably less extravasation of protein tracers as compared to the untreated group. Cell changes and edema at the ultrastructural level were mainly absent. Our results suggest that serotonin and prostaglandins are involved in heat stress induced breakdown of the BBB permeability, edema formation, and cell damage.