Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: Astroglial response and metallothionein expression

doi:10.1016/S0306-4522(99)00022-6

Neuroscience

Volume 92, Issue 3, June 1999, Pages 827-839

https://doi.org/10.1016/S0306-4522(99)00022-6 Get rights and content

Abstract

In this study we have evaluated the primary astroglial reactivity to an injection of N-methyl-d-aspartate into the right sensorimotor cortex, as well as the secondary astroglial response in the thalamic ventrobasal complex, caused by the anterograde degeneration of descending corticothalamic fibres and/or target deprivation of the developing thalamic neurons. The astroglial response was evaluated from 4 h to 30 days post-lesion, by the immunocytochemical detection of the cytoskeletal proteins glial fibrillary acidic protein and vimentin, and the antioxidant and metal binding protein metallothionein I–II. In the lesioned cortex, hypertrophied reactive astrocytes showed increased glial fibrillary acidic protein labelling that correlated with a strong expression of vimentin and metallothionein I–II. Maximal astrocytic response was seen at one week post-lesion. The glial scar that formed later on remained positive for all astroglial markers until the last survival time examined. In contrast, in the anterogradely/retrogradely affected thalamus, the induced astroglial secondary response was not as prominent as in the cortex and was characteristically transitory, being undetectable by 14 days post-lesion. Interestingly, thalamic reactive astrocytes showed increased glial fibrillary acidic protein expression but no induction of vimentin and metallothionein I–II.

In conclusion, in the young brain, the pattern of astroglial reactivity is not homogeneous and is strongly dependent on the grade of tissue damage: both in response to primary neuronal death and in response to retrograde/anterograde secondary damage, reactive astrocytes show hypertrophy and increased glial fibrillary acidic protein expression. However, astroglial vimentin and metallothionein I–II expression are only observed in areas undergoing massive neuronal death, where glial scar is formed.

Section snippets

Excitotoxic lesions

Long–Evans black-hooded nine-day old rats of both sexes (day of birth equals day 0) (Harlan Sprague–Dawley) were used in this study. Under ether anaesthesia, each pup was placed in a stereotaxic frame adapted for newborns (Kopf) and the skull was opened using a surgical blade. Thirty-seven nanomoles of N-methyl-d-aspartate (NMDA; Sigma, M-3262) diluted in 0.15 μl of saline solution (0.9% NaCl) were injected into the right sensorimotor cortex at the level of the coronal suture (2 mm lateral from

Results

The NMDA injection into the right sensorimotor cortex of immature rats induced neuronal degeneration accompanied by an astroglial response in the whole thickness of the cortex at the level of the injection site and surrounding tissue. In addition, an astrocytic response was observed in the VB complex of the thalamus. This thalamic response was likely to be due to secondary retrograde/anterograde changes. No astroglial response was seen in the contralateral hemisphere at any survival time.

Discussion

The present study describes a marked astroglial reaction in response to a neocortical excitotoxic lesion induced in nine-day-old rats. In the cortical injection site, the astroglial response is characterized by astrocyte hypertrophy, increased expression of GFAP and de novo expression of the intermediate filament vimentin and the metal binding protein metallothionein. Also, a characteristic astroglial reactivity was seen in the VB thalamic nuclei. This thalamic astrocytic reaction was

Conclusions

Excitotoxic lesions in the developing sensorimotor cortex are accompanied by a strong astroglial response first observed at one day after the injection. Hypertrophied reactive astrocytes show increased GFAP labeling and express vimentin and the metal binding protein metallothionein from day three post-injection. Maximal astrocytic hypertrophy is seen at day seven, and maximal GFAP, vimentin and metallothionein immunoreactivity is maintained at longer times, owing to glial scar formation. In

Acknowledgements

We are very grateful to M. A. Martil for the excellent technical assistance. This work was supported by DGICYT PB95-0662 project, CICYT SAF96-0189, PSPG PM-98-0170 and fellowships to L.A. from `la Caixa' Graduate Program and the F.P.I. Fellowship Program of the Universita Autònoma Barcelona.

References (76)

L. Acarin et al.
Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain
Expl Neurol.
(1997)
L. Acarin et al.
Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain. Microglial/macrophage response and MHC class I and II expression
Neuroscience
(1999)
K. Cowan et al.
Metallothionein induction by bismuth in neonatal rat primary astrocyte cultures
Brain Res.
(1996)
T. Dalton et al.
Temporalspatial patterns of expression of metallothionein-I and -III and other stress related genes in rat brain after kainic acid-induced seizures
Neurochem. Int.
(1995)
M. Ebadi et al.
Expression and regulation of brain metallothionein
Neurochem. Int.
(1995)
M. Ebadi et al.
The antioxidant properties of zinc and metallothionein
Neurochem. Int.
(1996)
M. Eddleston et al.
Molecular profile of reactive astrocytes—implications for their role in neurologic disease
Neuroscience
(1993)
S.A. Frautschy et al.
Localization of basic fibroblast growth factor and its mRNA after CNS injury
Brain Res.
(1991)
J. Hernandez et al.
Transgenic expression of interleukin-6 in the central nervous system regulates brain metallothionein-I and -III in mice
Molec. Brain Res.
(1997)
D.G. Herrera et al.
Glial fibrillary acidic protein immunoreactivity following cortical devascularizing lesion
Neuroscience
(1992)

J. Hidalgo et al.

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

Chem. Biol. Interact.

(1994)

O. Isacson et al.

Astroglial response in the excitotoxically lesioned neostriatum and its projection areas in the rat

Neuroscience

(1987)

K. Janeczko

The proliferative response of astrocytes to injury in the neonatal brain. A combined immunocytochemical and autoradiographic study

Brain Res.

(1988)

K. Janeczko

Co-expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere—a combined autoradiographic and double immunocytochemical study

Int. J. dev. Neurosci.

(1993)

K. Janeczko

Age-dependent changes in the proliferative response of S-100 protein-positive glial cells to injury in the rat brain

Int. J. dev. Neurosci.

(1994)

M.B. Jensen et al.

Morphological and immunophenotypic microglial changes in the denervated fascia dentata of adult rats: correlation with blood-brain barrier damage and astroglial reactions

Expl Neurol.

(1997)

B. Kolb et al.

Neonatal frontal cortical lesions in rats alter cortical structure and connectivity

Brain Res.

(1994)

B. Kolb et al.

Recovery from early cortical damage in rats. 7. Comparison of the behavioural and anatomical effects of medial prefrontal lesions at different ages of neural maturation

Behav. Brain Res.

(1996)

H. Konno et al.

Wallerian degeneration induces Ia-antigen expression in the rat brain

J. Neuroimmunol.

(1989)

S.W. Levison et al.

Acute exposure to CNTF in vivo induces multiple components of reactive gliosis

Expl Neurol.

(1996)

A. Logan et al.

Transforming growth factor-β1 and basic fibroblast growth factor in the injured CNS

Trends pharmac. Sci.

(1993)

T. Miyake et al.

Reactive proliferation of astrocytes studied by immunohistochemistry for proliferating cell nuclear antigen

Brain Res.

(1992)

G. Moonen et al.

Neurono-glial interactions and neural plasticity

Prog. Brain Res.

(1990)

M.M. Oblinger et al.

Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury

Int. J. dev. Neurosci.

(1993)

C.K. Petito et al.

Relationship between ischemia and ischemic neuronal necrosis to astrocyte expression of glial fibrillary acidic protein

Int. J. dev. Neurosci.

(1993)

S.K.R. Pixley et al.

Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin

Devl Brain Res.

(1984)

M. Sato et al.

Oxygen free radicals and metallothionein

Free Radic. Biol. Med.

(1993)

D. Schiffer et al.

Immunohistochemistry of glial reaction after injury in the rat—double stainings and markers of cell proliferation

Int. J. dev. Neurosci.

(1993)

J.P. Schwartz et al.

Neurotrophic factor gene expression in astrocytes during development and following injury

Brain Res. Bull.

(1994)

P.A. Sillevis Smitt et al.

Metallothionein immunoreactivity is increased in the spinal cord of patients with amyotrophic lateral sclerosis

Neurosci. Lett.

(1992)

G.M. Smith et al.

Immature type-1 astrocytes suppress glial scar formation, are motile and interact with blood vessels

Brain Res.

(1991)

C. Steinhauser et al.

News on glutamate receptors in glial cells

Trends Neurosci.

(1996)

Y. Takamiya et al.

Immunohistochemical studies on the proliferation of reactive astrocytes and the expression of cytoskeletal proteins following brain injury in rats

Brain Res.

(1988)

Y. Uchida et al.

The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein

Neuron

(1991)

J.M. Vela et al.

Induction of metallothionein in astrocytes and microglia in the spinal cord from the myelin-deficient jimpy mouse

Brain Res.

(1997)

M.N. Woodroofe et al.

Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in vivo microdialysis: evidence of a role for microglia in cytokine production

J. Neuroimmunol.

(1991)

L. Acarin et al.

Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified microglial cells in rat brain by tomato lectin binding

J. Histochem. Cytochem.

(1994)

C. Aoki et al.

Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats

J. Neurosci.

(1994)

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Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: Astroglial response and metallothionein expression

Abstract

Section snippets

Excitotoxic lesions

Results

Discussion

Conclusions

Acknowledgements

Expl Neurol.

Neuroscience

Brain Res.

Neurochem. Int.

Neurochem. Int.

Neurochem. Int.

Neuroscience

Brain Res.

Molec. Brain Res.

Neuroscience

Chem. Biol. Interact.

Neuroscience

Brain Res.

Int. J. dev. Neurosci.

Int. J. dev. Neurosci.

Expl Neurol.

Brain Res.

Behav. Brain Res.

J. Neuroimmunol.

Expl Neurol.

Trends pharmac. Sci.

Brain Res.

Prog. Brain Res.

Int. J. dev. Neurosci.

Int. J. dev. Neurosci.

Devl Brain Res.

Free Radic. Biol. Med.

Int. J. dev. Neurosci.

Brain Res. Bull.

Neurosci. Lett.

Brain Res.

Trends Neurosci.

Brain Res.

Neuron

Brain Res.

J. Neuroimmunol.

Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified microglial cells in rat brain by tomato lectin binding

J. Histochem. Cytochem.

Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats

J. Neurosci.