Manuel Nieto-Sampedro

Transplants of purified astrocytes promote behavioral recovery after frontal cortex ablation

Ablation of the medial frontal cortex produces a learning deficit on a reinforced alternation task. Recovery from this deficit was significantly accelerated in rats by transplantation of either cultured purified astrocytes or Gelfoam that had remained the previous 5 days in a brain wound in another animal (wound-Gelfoam). Cell-free extracts of wound-Gelfoam did not enhance behavioral recovery. Embryonic frontal cortex was effective only if transplanted with a delay after ablation. It appears from these results that transplants can facilitate functional recovery by more than one mechanism, including promotion of survival and reactive synaptogenesis of host neurons, stabilization of the damaged environment and replacement of lost neurons. In this study, glial cells were capable of facilitating recovery from central nervous system damage to the same extent as neuronal transplants.

Induction of a neurite-promoting factor in rat brain following injury or deafferentation

Ablation of the entorhinal/occipital cortex in young adult rats caused a several-fold increase in the neurite-promoting activity in extracts of the tissue surrounding the wound and in areas that had been deafferented by the lesion. The time course of induction closely paralleled reactive axon sprouting in the deafferented hippocampus, with maximal levels of neurite-promoting activity reached between 9 and 15 days post-lesion. Aged animals, in which reactive sprouting is deficient, showed no increase in activity by 12 days after deafferentation of the hippocampus. The neurite-promoting activity of brain extracts was non-diffusible, heat-labile, and sensitive to proteolysis. All of the activity bound to diethylaminoethyl (cellulose) and was eluted at 200 mM NaCl. The apparent molecular weight (by gel filtration) of the activity in extracts of uninjured brain was 9-17 kilodaltons, whereas the extracts of injured brain also had peaks or shoulders at 30, 70 and greater than or equal to 200 kilodaltons. These data suggest that the brain neurite-promoting activity resides in one or more proteins. Both the injury-induced and basal activities were different from laminin, nerve growth factor, and polyornithine-bindable neurite-promoting factors. The injury-induced activity was sensitive to repeated freezing and thawing, but this inactivation was reversed by thiol reagents such as glutathione, thioglycerol, and mercaptoethanol. We report a neurite-promoting factor that is induced following brain injury or denervation, and may also be important for reactive axon sprouting after brain injury. The induction of this factor is abnormal in aged animals, as is the reactive sprouting response. The properties of the injury-induced activity distinguish it from the basal activity (found in uninjured brain) and from other characterized neurite-promoting factors.

Neuronotrophic activity in brain wounds of the developing rat. Correlation with implant survival in the wound cavity.

Neuronotrophic activity accumulates in a wound cavity created in the entorhinal/occipital cortex of developing rats. These trophic factors support the survival of neurons in monolayer cultures of chick embryo spinal cord, ciliary ganglion, sympathetic ganglion and dorsal root ganglion, as well as of mouse dorsal root ganglion. Trophic activity was very low both in non-injured brain tissue and in the wound cavity 1 day post-lesion, but it increased 15- to 300-fold during the subsequent 2-5 days. Together with the trophic activity in the wound fluid were other substances which interfered with the survival of spinal cord neurons. The neuronotrophic factors appeared to be proteins immunologically distinct from mouse submaxillary nerve growth factor. Fragments of rat embryo corpus striatum placed in the cortical wound cavity immediately after its formation showed very poor subsequent survival and no innervation of the host hippocampus. However, if implantation was delayed by 3 or 6 days with respect to the time at which the receiving cavity was made, the survival was greatly improved and innervation of the host took place. The time course for the accumulation of the trophic factors in the cavity paralleled the delay leading to increased survival of brain grafts. It is suggested that the neuronotrophic activity accumulating in the wound cavity during the delay period may be responsible for the increased survival of the implants.

Epidermal growth factor receptor immunoreactivity in rat brain. Development and cellular localization

In rat brain, distinct epidermal growth factor-receptor immunoreactivity (EGFR-IR) first appeared in astroglia at about day 16 postnatal, reached maximum intensity at 19 days and then became much weaker as the animals reached adulthood. EGFR-IR was also observed in cerebellar Purkinje cells as early as 11 days postnatal and was maintained into adulthood. In adult and aged animals the most prominent EGF receptor immunostaining occurred in cerebral cortex neurons (layers IV and V) that had the morphology of basket cells. Immunoreactive neurons were abundant in the cingulate, frontal, frontoparietal and striate cortices. In the frontoparietal cortex, EGFR positive neurons were most numerous in the motor area, diminishing laterally towards the somatosensory area. The localization and time of appearance of EGFR-IR did not appear consistent with a direct mitogenic role of the EGF domain in astroglia proliferation during development. However, the EGFR may be involved in neuronal survival and/or neuron-glia signalling.

The control of glial populations in brain: Changes in astrocyte mitogenic and morphogenic factors in response to injury

Injury to rat brain induces a 3-10-fold increase in the activity of factors capable of stimulating astrocyte DNA synthesis and cell division in vitro. Maximum mitogenic activity was reached 10-15 days post-lesion in both the tissue surrounding the wound and in the gelfoam filling the wound cavity. Factors capable of transforming the astrocyte morphology from polygonal-flat to fibrous-like (morphogens) could also be observed in brain tissue and showed increased activity beginning at 10 days postlesion. On the other hand, morphogenic activity was very low or absent in gelfoam extracts until 15 days postlesion. Both mitogenic and morphogenic factors were nondiffusible and were partly temperature and trypsin sensitive, i.e. they had the properties of protein-like substances, but seemed different from both epidermal and fibroblast growth factors. As judged by their filtration behavior on Amicon membranes, the molecular weight of mitogens and morphogens ranged from lower than 30,000 to greater than 100,000. Inhibitors of both mitogenic and morphogenic activities with molecular weight lower than 30,000 seemed to be also present in the brain extracts. The factors described here can account for the processes of astrocytosis and astrogliosis observed in vivo in response to CNS injury.

Neuronotrophic factors for mammalian brain neurons: injury induction in neonatal, adult and aged rat brain.

Tissue from neonatal, adult and aged Sprague-Dawley rat brain contained low levels of survival promoting activity for embryonic neurons from various rat brain regions. This basal neuronotrophic activity was 2-fold higher in adult and 4-fold higher in aged rat brain, with respect to that in neonatal brain. Tissue extracts also contained 4-fold higher levels of neuronotoxic activity in adult and aged than in neonatal brain. Ablation of the occipital/entorhinal cortex caused a 3-fold increase in neuronotrophic activity in the tissue surrounding the wound in neonates and 4-fold in adult and aged brain, with respect to the basal levels. Maximal activities occurred at 3 days, 6 days and 15 days postlesion in neonatal, adult and aged tissue respectively, and subsequently returned to basal levels. Neuronotoxic activity was not induced following injury. The slower neuronotrophic response to injury in older animals may be one of the determinant factors of the slower recovery from brain damage observed in aging.

Epidermal growth factor receptor immunoreactivity in rat brain astrocytes. Response to injury

Brain astroglia in normal adult rats stained weakly or not at all with an antibody to epidermal growth factor receptor (EGFR). A dramatic change took place after injury. The astrocytes adjacent to an entorhinal ablation and in deafferented areas of the hippocampus showed prominent EGFR immunoreactivity. Cells that were EFGR-immunoreactive also stained intensely with an antibody to glial fibrillary acidic protein (GFAP). The localization and the time course of appearance of EGFR/GFAP immunoreactivity suggests that EGFR may be involved in the conversion of a normal into a reactive astrocyte.

Neuroprotective effect of MK-801 and U-50488H after contusive spinal cord injury

One hour before a contusive spinal cord injury either compound MK-801 or compound U-50488H was injected intraperitoneally, and a 14-day-delivery osmotic minipump containing the same drug was placed subcutaneously at the time of surgery. The motor and sensory behavior of the animals was measured over the following 30 days. Both MK-801 and U-50488H treatments had a statistically significant neuroprotective effect. The number of neurons per unit area outside the lesion epicenter was significantly (P less than 0.01) greater in the drug-treated animals (MK-801, 298.9 +/- 74.8 neurons/mm2; U-50488H, 242.7 +/- 16.5 neurons/mm2) than in untreated controls (73.3 +/- 9.3 neurons/mm2). Recovery of sensory and motor behavior was limited but significant differences were observed when drug-treated rats were compared with untreated controls. The effects of the two drugs were not additive for any of the variables studied.

Early release of glia maturation factor and acidic fibroblast growth factor after rat brain injury

A major component of the healing response of the brain to injury is the induction of growth and trophic factors. In the rat brain, glia maturation factor (GMF) and acidic fibroblast growth factor (aFGF) are not extracellular. However, within the first hour following brain injury, the amount of GMF and aFGF in the wound cavity increased by 7- and 13-fold, respectively, compared to the tissue adjacent to the wound. A cascade of cellular and biochemical events, leading to glial proliferation, the arrest of secondary neuronal death and axonal sprouting, may be initiated by the sudden increase in the extracellular concentration of these factors.

Neuronotrophic activity for ciliary ganglion neurons. Induction following injury to the brain of neonatal, adult, and aged rats

The induction of neuronotrophic activity following injury to the brain of neonatal, adult, and aged Sprague-Dawley rats was compared using an improved in vitro assay. The maximal levels of activity in tissue surrounding the wound were reached at 3, 10-15, and about 15 days postlesion in neonatal, adult, and aged animals, respectively. Tissue neuronotrophic levels were always much lower in neonatal animals relative to the older animals. Accumulation of neuronotrophic activity in the gelfoam placed into the wound cavities in neonatal and adult animals lagged behind the levels in tissue by 4-5 days, suggesting that either the neuronotrophic factor itself or the cells which produce it are transferred from the tissue into the gelfoam. Relatively little activity accumulated in the gelfoam taken from aged Sprague-Dawley rats, and this observation was confirmed in aged Fischer rats. Aged animals seem to be unable to produce or release one of a number of neuronotrophic factors in response to injury.