Isolation of rat lung mast cells for purposes of one-week cultivation using novel Percoll variant Percoll PLUS.

Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative "asymptotically isotonic" stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasting cultivation and observed some model morphological changes of LMC.

In vitro hypoxia increases production of matrix metalloproteinases and tryptase in isolated rat lung mast cells.

Chronic hypoxia results in hypoxic pulmonary hypertension characterized by fibrotization and muscularization of the walls of peripheral pulmonary arteries. This vessel remodeling is accompanied by an increase in the amount of lung mast cells (LMC) and the presence of small collagen cleavage products in the vessel walls. We hypothesize that hypoxia activates LMC, which release matrix metalloproteinases (MMPs) cleaving collagen and starting increased turnover of connective tissue proteins. This study was designed to determine whether in vitro hypoxia stimulates production of MMPs in rat LMC and increases their collagenolytic activity. The LMC were separated on the Percoll gradient and then were divided into two groups and cultivated for 24 h in 21 % O(2) + 5 % CO(2) or in 10 % O(2) + 5 % CO(2). Presence of the rat interstitial tissue collagenase (MMP-13) in LMC was visualized by immunohistological staining and confirmed by Western blot analysis. Total MMPs activity and tryptase activity were measured in both cultivation media and cellular extracts. Exposure to hypoxia in vitro increased the amount of cells positively labeled by anti-MMP-13 antibody as well as activities of all measured enzymes. The results therefore support the concept that LMC are an important source of increased collagenolytic activity in chronic hypoxia.

Conditioned blocking is re-established by neurotransplantation in mature rats.

Aging has been associated with a decay of hippocampal function that may begin well before senescence. Conditioned blocking is a complex learning phenomenon that requires an intact hippocampus in young-adult rats and is absent in middle-aged rats. The aim of the present study was to test the possibility of re-establishing conditioned blocking in 17-month-old Wistar rats by neurotransplantation. Solid embryonic hippocampal or nigral tissue was bilaterally transplanted in the proximity of the dorsal hippocampus (lateral ventricle and alveus). Conditioned blocking of an aversion to a cider vinegar (3%) solution presented in compound with a previously conditioned saccharin solution (0.1%) appeared 14 days after transplantation and persisted 3 months later only in the hippocampal grafted group, showing the possibility of restoring age-related cognitive deficits.

Retinal ganglion cells regenerating through the peripheral nerve graft retain their electroretinographic responses and mediate light-induced behavior.

To assess the light-induced electrical activity of rodent retinal ganglion cells (RGCs) regenerating into a peripheral nerve (PN) graft we used non-invasive recording of electroretinographic responses to the contrast-reversal of sinusoidal gratings (p-ERG). On comparing the retinas that received a PN graft and retinas with only optic nerve (ON) transection, p-ERG responses were present in grafted retinas as late as 20 months after the surgery while they completely disappeared in non-transplanted controls within 4 months of ON transection. Next, the ability of regenerating RGCs to form functional connections with their targets in the superior colliculus (SC) was tested by a light-escape task. While the bilaterally blinded animals did not improve during the test, unilaterally grafted animals (with the contralateral eye blinded) reached 26% success in the last quartile of the light-escape task. This performance was significantly better than that of blind animals (ANOVA and Student-Newman-Keuls test; p<0.05), but did not reach the level of intact rats (87%). The transplanted rats, therefore, were capable of light perception, but at a sub-normal ability. In addition, we were also able to correlate the amplitude of the p-ERG response with the visual behavioral performance for each transplanted animal. This finding indicates that there is a direct link between the RGC electrophysiological activity and the functional capacity of the regenerated visual pathway. In conclusion, the above results indicate that (a) PN grafts help to preserve the normal electroretinographic activity of injured and regenerating RGCs (b) the regenerated visual pathway is functional and capable of mediating simple visual behavior and that (c) there is a correlation between the light-evoked RGC electrical activity and visual behavior and, finally, that (d) the effect of PN graft on the electrophysiological and functional restoration of the visual pathway is long-lasting or even permanent.

Effects of NGF, b-FGF, and cerebrolysin on water maze performance and on motor activity of rats: short- and long-term study.

The effects of 14-day treatments with nerve growth factor (NGF), basic fibroblast growth factor (b-FGF), or the peptidergic drug Cerebrolysin on postlesion acquisition of a water maze task and on motor activity were evaluated. Rats were tested in the Morris water maze 14 days (early test) and 7 to 8 months (delayed test) after a bilateral lesion of the frontoparietal (sensorimotor) cortex. Only the rats treated with Cerebrolysin performed the water maze task at the level of the nonlesioned controls in the early test. No short-term effect of NGF (6.5 ng/14 days; 38 ng/ml) or b-FGF (17 ng/14 days; 100 ng/ml) treatment was found. The delayed test revealed that water maze performance was restored in rats treated with b-FGF in comparison with intact controls. The data showed that b-FGF can support or initiate processes in the CNS that lead to a delayed functional amelioration and/or compensation for a water maze performance deficit. NGF did not influence the acquisition impairment caused by a sensorimotor cortical lesion. Two-week administration of Cerebrolysin had a time-dependent influence: it attenuated the acquisition deficit and increased the motor activity of rats, both effects declined to the level of lesioned controls within 8 months.

Can Cerebrolysin influence chronic deterioration of spatial learning and memory?

Our previous studies showed that the nootropic drug Cerebrolysin, applied immediately after the traumatic or excitotoxic brain lesion influenced spatial learning and memory. Long-lasting ameliorative effect of Cerebrolysin was found after its 4-week-administration, while two-week-treatment had only temporal effect. With the aim to verify the capability of Cerebrolysin to restore chronically deteriorated learning and memory. The drug was applied 4 months after lesioning the rat's CNS. The present study shows that Cerebrolysin restored learning capability of the lesioned rats. Although their spatial memory was improved in comparison to lesion untreated controls, it did not reach the level of intact controls. The effect was more pronounced after the application of 1.25 ml/kg b. w. of Cerebrolysin than after the application of 2.5 ml/kg b. w.

Fetal hippocampal transplants restore conditioned blocking in rats with dorsal hippocampal lesions: effect of age.

Previous studies have shown that conditioned blocking of taste aversion learning in 3-month-old Wistar rats depends on the hippocampal system integrity. Thus, the aim was to demonstrate that enough connectivity would develop after a graft to support the attention mechanism required for conditioned blocking. In the first experiment, bilateral homotopic grafts of 16-17 day-old hippocampal fetal tissue applied to 3-month-old male Wistar rats after electrolytical lesions of the dorsal hippocampus reinstated conditioned blocking tested 5 months after the transplantation. Unexpectedly, an early age-dependent impairment of conditioned blocking, similar to that induced by hippocampal lesions, was found in the 8-month-old control group. This finding was further supported by the results of the second experiment. Non-operated 3-month-old but not 8-month-old Wistar rats showed conditioned blocking. The results are discussed in terms of early hippocampal vulnerability, prevented by fetal grafts.

Ameliorative influence of a nootropic drug on motor activity of rats after bilateral carotid artery occlusion.

The effects of the peptidergic nootropic drug Cerebrolysin on spatial memory and motor activity were examined in intact and ischemic rats. Ischemic-hypoxic damage was induced by injection of Na-cyanide followed by bilateral occlusion of common carotid arteries. Immediately afterwards Cerebrolysin or saline was administered, either by continuous intraventricular (i.v.) infusion or by daily intraperitoneal (i.p.) injection. Rats were tested for spatial memory and motor activity in the Morris water maze on days 3 and 4 post-surgery. The best dose of the substance for i.p. administration was known from previous studies. Therefore we had to investigate the dose-response-relationship and tolerability of the drug after i.v. administration in intact rats. Infusion (i.v.) of a high dose of Cerebrolysin (0.57 mg/day) decreased motor activity and spatial memory of intact rats (p < 0.01 and p < 0.05, respectively) but low dose of Cerebrolysin was well tolerated in the intact animals. Ischemia led to deterioration of motor activity in control rats (p < 0.01). Cerebrolysin significantly counteracted deleterious motor changes due to ischemia up to the level of intact controls after both i.v. infusion (0.0057 mg/day) and daily i.p. drug administration (100 mg/kg bw and day) indicating an accelerating recovery after ischemia.

Nerve growth factor and nootropic drug Cerebrolysin but not fibroblast growth factor can reduce spatial memory impairment elicited by fimbria-fornix transection: short-term study.

In an attempt to compare effects of different neurotrophic factors on impaired memory function, young adult naive rats were trained to find the hidden platform in the Morris water maze (3 consecutive days, eight trials/day). The fimbria-fornix was unilaterally removed by aspiration and nerve growth factor (NGF) (11 micrograms/ml and 0.5 microgram/ml; groups NGF and ngf, respectively) or basic fibroblast growth factor (bFGF) (0.2 microgram/ml, group FGF) were applied via intra-cerebroventricular infusion by the osmotic minipump (flow rate 0.5 microliter/h, 14 days). Nootropic drug Cerebrolysin (EBEWE Arzneitmittel; 2.5 ml/kg/day, group CER) was applied via intraperitoneal injection (14 days). One group was formed by the rats treated with NGF (11 micrograms/ml) and Cerebrolysin (group NGFCER). Non-lesioned and lesioned only rats served as controls (groups INT and LES). After a 14-day treatment, rats were tested using the retention test (1 day, four trials). On the next day, the rats were tested using transfer test (3 days, eight trials/day). Escape latency and length of trajectory was recorded. Groups NGF, ngf, FGF and LES were similarly impaired in their ability to retrieve the old position of the platform (retention test), as well as in their ability to navigate to the new position of the platform (transfer test). In the latter, NGF group significantly differed from lesioned animals. Groups CER and NGFCER were comparable to group INT in the retention or transfer test. It is concluded that anterograde amnesia elicited by fimbria-fornix lesion can be abbreviated by NGF and/or CER, while retrograde amnesia is absent only in rats treated by CER. No short-term influence of bFGF was found. It is suggested that biochemical systems other than the cholinergic one are involved.

Unilateral grafting of fetal neocortex into a cortical cavity improves healing of a symmetric lesion in the contralateral cortex of adult rats.

Fetal neocortical tissue (ED 14) was grafted unilaterally into a cortical cavity made bilaterally in the sensorimotor cortex of adult rats. Transplantation was done immediately after the lesion (group TR0, n = 8) or with 14-day delay (group TR14, n = 8). Six rats served as lesion only controls (group LES). After long-term survival (up to 15 months) the brains were photographed and surface areas of transplant and contralateral cavities were measured by means of a graphic tablet. The results show that (a) the presence of a transplant in one lesion cavity in the cortex decrease the size of a similar cavity in the contralateral cortex and that (b) the better host transplant integration there is, the greater the effect on the contralateral lesion. No correlation between the size of the transplant and the size of the symmetric traumatic lesion was found. The ameliorating effect of the transplant on the contralateral cortical lesion size is most likely related the long-term influence of growth of trophic factors released by transplanted cells which lead to the healing of the symmetric lesion.

Integration of neocortical embryonal grafts with the neocortex of host rats examined by Leao's spreading cortical depression.

Cortical spreading depression (SD) was used to assess the density and organization of neural elements in neocortical transplants and their connectivity with the host brain. Embryonal neocortex (E14) was transplanted into cavities in the frontoparietal cortex of 3-month-old rats. SD elicited in the cortex of anesthetized host rats (n = 12) 3 to 8 months after transplantation did not penetrate into the grafts. SD could be elicited in large transplants but did not propagate to the surrounding host neocortex. Spontaneous unit activity in the transplants was affected by SD elicited in the neocortex of the host rats anesthetized with urethane. Most units (n = 49) displayed excitatory-inhibitory (52%) or inhibitory (29%) reactions, whereas purely excitatory reactions were less frequent (8%). The results suggest that the packing density of neurons in the transplant can support SD but that the conditions at the graft-host boundary (glial scar, scarcity of neurons) stop SD propagation. High reactivity of the graft neurons to SD in the host neocortex indicates that afferentation from the host brain represents an important, predominantly excitatory contribution to the spontaneous activity of the transplant.

The influence of mechanical injury on the metabolic activity of transplanted cerebral cortex.

The authors studied the metabolic activity of rat embryonic cerebral cortex grafts (ED 15-16) implanted into rat brains immediately (TR0) and 14 days (TR14) after cavity formation. Over a period of two months, the ATP, lactate and glucose concentration in TR0 transplants remained at the same level as observed in the intact cortex, whereas in TR14 transplants the ATP and glucose concentration fell significantly and the lactate concentration rose. The DNA concentration rose in both types of transplants, but the increase was more pronounced in TR0 grafts. Choline acetyltransferase activity (a neuron marker) fell significantly in both cases, but the decrease was greater in TR14 transplants. The results indicate that grafts implanted into the brain immediately after cavities had been formed have better metabolic activity and are capable of longer survival than grafts implanted 14 days after cavitation.

Unilateral striatal grafts induce behavioral and electrophysiological asymmetry in rats with bilateral kainate lesions of the caudate nucleus.

Rats (n = 11) with bilateral kainate lesions of the caudate nucleus and subsequent unilateral transplantation of embryonic striatal tissue into the damaged area preferred 4 months later to reach for food with the forepaw contralateral to the graft. No such asymmetry was observed in lesioned, nontransplanted (n = 8) or unoperated (n = 5) control rats. Good integration of the graft with the host brain was indicated by the finding that cortical spreading depression did not enter the lesioned caudate nucleus but did penetrate into the lesioned caudate with the graft almost as regularly as in intact rats. Behavioral asymmetry produced by unilateral grafts in bilaterally lesioned animals reveals the effects of transplantation with more sensitivity than the graft-induced compensation of the asymmetries caused by unilateral lesions.

The morphological picture of implanted foetal rat brain tissue.

The authors studied the morphological picture of implanted foetal brain tissue. Macroscopically, they found that the grafts used for morphological study had taken in every case and that the size of the graft had doubled or trebled. The surface of the implant grew above the level of the recipient's cortex and numerous branching blood vessels were observed in the graft-cortex contact area. In the light microscope, organization of the cells in circular clusters or strips was found in the implant. Large numbers of blood vessels of varying calibre were present in the vicinity of large cell concentrations. Distinct differences between the cytoarchitectonics of the normal tissue of the recipient and the graft tissue were seen. Cells with a normal appearance or an apolar form, whose nucleus contained irregularly scattered chromatin and mosaic-like nucleoli, were observed at ultrastructural level. The nuclear membrane was thrown into multiple folds which invaginated deep into the nuclear matter. The Golgi complex covered a large area. Axodendritic synapses indistinguishable from the control were found in the neuropil. No changes were observed in the walls of the capillaries. The pericapillary zone was characterized by the presence of light astrocyte processes.

Development of Na, K-ATPase in neocortical grafts.

Pieces of cerebral cortex from 14-day rat embryos were transplanted into freshly prepared cavities in the cerebral cortex of adult rats. At various time intervals after implantation. Na,K-ATPase, Mg-ATPase as well as the ratio of two molecular forms of Na,K-ATPase were determined in the grafts and compared with the values obtained from intact cortex at various stages of postnatal development. Up to the 57th postimplantation day (PID), Na.K-ATPase develops very slowly, reaching on PID 57, i.e. on postconceptional day (PCD) 71, only 40% of the enzyme activity of intact brain cortex (PND 49, PCD 71). At PID 90 (PCD 104) Na,K-ATPase activity attained that of the intact adult tissue (PND 82, PCD 104). Mg-ATPase activity in the grafts developed similarly to that of intact brain but was much higher in the early postimplantation phase. The maximum ratio of the two molecular forms of Na,K-ATPase [alfa(+)/alfa] was shifted toward later developmental periods in the grafts as compared with the intact cerebral cortex.