Entorhinal Cortex Lesion with bFGF Analysis — F Gomez-Pinilla | ReplicateScience

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Entorhinal Cortex Lesion with bFGF Analysis — F Gomez-Pinilla | ReplicateScience

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Basic fibroblast growth factor (bFGF) is a potent trophic factor for neurons and astrocytes and recently has been implicated in the pathology of Alzheimer's disease. In order to better understand the role of bFGF in normal brain function and during pathology, we have analyzed its anatomical distribution and its response to injury in the CNS. Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations, and occasionally in microglial cells throughout the normal rat brain. Neuronal populations that showed bFGF immunoreactivity included septohippocampal nucleus, cingulate cortex, subfield CA2 of the hippocampus, cerebellar Purkinje cells, cerebellar deep nuclei, facial nerve nucleus, and the motor and spinal subdivisions of the trigeminal nucleus and facial nerve nucleus. The pattern of bFGF immunoreactivity in the hippocampus was examined following entorhinal cortex lesion, or fimbria-fornix transection. After entorhinal cortex lesion, bFGF immunoreactivity increased in the outer molecular layer of the dentate gyrus ipsilateral to the lesion. The lesion effect on bFGF immunoreactivity was expressed as an increase in the number of bFGF astrocytes, as an increase in the intensity of bFGF immunoreactivity within astrocytes, and as an increase of bFGF immunoreactivity in the surrounding extracellular matrix, relative to the contralateral side. The time course and pattern of reorganization paralleled the sprouting of septal cholinergic terminals in response to the same type of lesion, suggesting that bFGF may play an important role in lesion-induced plasticity. After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex and, as previously reported, prevent the death of medial septal neurons. Therefore, it appears that bFGF infusion, which has been shown to increase the synthesis of NGF by astrocytes (Yoshida and Gage, 1991), also helps enable neurons to respond to NGF. This suggests that after injury bFGF may participate in a cascade of neurotrophic events, directly and indirectly facilitating neuronal repair and/or promoting neuronal survival.

1

Tissue preparation and sectioning

Prepare rat brain tissue for immunohistochemical analysis

not specifiednot specified

Note: Tissue from normal adult rats and lesioned animals

View evidence from paper

“Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations”

2

Double-staining immunohistochemistry

Perform double-staining immunohistochemistry to detect bFGF immunoreactivity and identify cell types

not specifiednot specified

Note: Allows simultaneous identification of bFGF in astrocytes, neurons, and microglial cells

View evidence from paper

“Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations, and occasionally in microglial cells”

3

Map bFGF distribution in normal brain

Document anatomical distribution of bFGF immunoreactivity throughout normal rat brain

not specifiednot specified

Note: Identify neuronal populations and brain regions with bFGF immunoreactivity

View evidence from paper

“Neuronal populations that showed bFGF immunoreactivity included septohippocampal nucleus, cingulate cortex, subfield CA2 of the hippocampus, cerebellar Purkinje cells”

4

Perform entorhinal cortex lesion

Create lesion of the entorhinal cortex in experimental animals

not specifiednot specified

Note: Lesion is unilateral; contralateral side serves as control

View evidence from paper

“The pattern of bFGF immunoreactivity in the hippocampus was examined following entorhinal cortex lesion”

5

Analyze bFGF response to entorhinal cortex lesion

Examine changes in bFGF immunoreactivity in hippocampus following entorhinal cortex lesion

not specifiednot specified

Note: Compare ipsilateral (lesioned side) to contralateral (control) side

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“After entorhinal cortex lesion, bFGF immunoreactivity increased in the outer molecular layer of the dentate gyrus ipsilateral to the lesion”

6

Quantify bFGF changes

Measure increases in bFGF immunoreactivity as number of bFGF astrocytes, intensity within astrocytes, and extracellular matrix immunoreactivity

not specifiednot specified

Note: Compare lesioned side to contralateral control side

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“The lesion effect on bFGF immunoreactivity was expressed as an increase in the number of bFGF astrocytes, as an increase in the intensity of bFGF immunoreactivity within astrocytes”

7

Perform fimbria-fornix transection

Create transection of the fimbria-fornix in separate cohort of experimental animals

not specifiednot specified

Note: Separate experimental group from entorhinal cortex lesion animals

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“After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors”

8

Chronic infusion of bFGF

Administer chronic infusion of bFGF following fimbria-fornix transection

chronic (duration not specified)not specified

Note: bFGF infusion preserves NGF receptors and prevents medial septal neuron death

View evidence from paper

“After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex”

9

Analyze NGF receptor preservation

Examine NGF receptor expression on neurons in medial septal complex following bFGF infusion

not specifiednot specified

Note: Assess neuronal survival in medial septal complex

View evidence from paper

“chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex and prevent the death of medial septal neurons”