88 B
astrocytes [31,50,63]. However, it is hypothesized that trocytosis, neoangiogenesis and VEGF expression from a
even moderate reactive astrocytosis may interfere with spectrum of conditions including Alzheimer’s disease,
eventual reconnection of functional neuronal circuitry by brain abscess, peritumoral brain from metastatic car-
inhibiting axonal regeneration, preventing remyelination or cinoma, head injury and cerebral infarcts. Further, to
promoting abnormal neuronal connections with increased evaluate the temporal relationship of reactive astrocytosis
seizure foci [57]. and neoangiogenesis, a stereotactic brain injury model was
There is currently little understanding of the molecular utilized. In order to obtain the proliferative index [ Ki67
mechanisms regulating reactive astrocytosis. Several ob- positive cells 1000 endothelial cells3100 of endothelial
servations suggest that reactive astrocytosis is a regulated cells, surgical specimens from brain abscess and metastatic
process involving cytokines, inflammatory mediators, carcinoma were stained for Ki-67. In addition, a CNS
growth factors and physiological stimuli such as hypoxia injury model was utilized in transgenic mice which express
[45,50,69]. For example, axonal growth and regeneration Green Fluorescent Protein GFP under the control of the
after trauma and cerebral infarction are retarded by inhib- astrocyte specific GFAP promoter [5], to co-localize VEGF
itory proteins associated with the intense astrogliotic scar expression in reactive astrocytes.
[6,45,50]. Second, reactive astrocytosis although most intense immediately surrounding the area of injury, is
found at much removed distances suggestive of soluble
2. Methods
trophic factors acting in a paracrine fashion [50]. While a variety of these trophic factors have been studied in
2.1. Evaluation of human neuropathological diseases astrocyte cell culture experiments, their role and interac-
tions in the astrogliotic response in vivo is poorly under- In order to evaluate neoangiogenesis and VEGF expres-
stood. Specifically, polypeptide growth factors such as sion in a variety of reactive astrogliotic specimens, 23
basic Fibroblast Growth Factor bFGF, Epidermal Growth cases of neurosurgical and autopsy specimens obtained
Factor EGF, Transforming Growth Factor TGF-a, from our neuropathology department were examined.
Platelet Derived Growth Factor PDGF, Cilliary Neuro- These cases represented a spectrum of benign and malig-
trophic Factor CNTF and cytokines such as Interleukin-1 nant diseases that are known to induce reactive astrocytosis
IL1 and Tumor Necrosis Factor TNF-a, have all been in the surrounding brain and included: four Alzheimer’s
implicated in
the astrogliotic
response [3,13,32,37,
Disease AD, three bacterial abscesses, five carcinomas, 49,50,59,68,69].
three head injuries and seven infarcts two acute: ,7 days, Part of any reparative tissue process is neoangiogenesis,
two subacute: 7–30 days, three old: .30 days. Normal vividly demonstrated in the collagen fibrovascular response
autopsy brain tissue was used as a control. Standard associated with wound healing and one that can be
hematoxylin and eosin HE staining was performed on speculated to be an important component of reactive
5-mm tissue sections from paraffin embedded tissue astrocytosis in the CNS [10]. Amongst the regulators of
blocks, which were independently reviewed by a neuro- neoangiogenesis, Vascular
Endothelial Growth
Factor pathologist PS. Sequential sections were incubated over-
VEGF is highly potent and specific by activating its night at 48C with a primary rabbit antibody against GFAP
cognate receptors on the endothelial cells resulting in their 1:3000, Dako Corp, CA to mark reactive astrocytes, a
proliferation, migration and sprouting [17,27,61]. VEGF human anti-Factor VIII antibody 1:2000, Dako Corp to
has been implicated in most physiological and pathological determine the microvascular density MVD or a polyclon-
processes involving neovascularization including embryo- al rabbit anti-VEGF antibody 1:50, Santa Cruz Biotech-
genesis, wound healing, tumor growth, myocardial is- nology Inc., CA that recognizes all four VEGF isoforms.
chemia, ocular neovascular diseases and chronic diseases Detection was through the ABC Vector-DAB VectaStain
such as rheumatoid arthritis [16,21]. In addition to its Elite, CA system. The sections were analyzed using the
angiogenic effects, VEGF is also a potent edemogenic MicroComputer Image Device MCID; Imaging Research
agent, resulting in disruption of the blood–brain-barrier Inc., Canada, linked to a color CCD camera Sony DXC
BBB, and hence was also known as Vascular Permeabili- 970 MD mounted on a transmitted-light microscope
ty Factor VPF. We observed that VEGF VPF was co- Zeiss Axioskop fitted with a Ludl Biopoint motorized
expressed with GFAP-positive reactive astrocytes in the stage. Microscope fields 4003magnification were ac-
peritumoral edematous brain around meningiomas, where quired and digitized for each of the sequential sections
its expression was correlated to the vascularity and associ- stained for GFAP, Factor VIII and VEGF, with 4–6 high-
ated edema induced by these CNS tumors [56]. This powered fields HPF analyzed in each tissue section.
suggested that reactive astrocytes may express VEGF, These HPFs were chosen to be adjacent to the site of tissue
modulating neoangiogenesis and also the edema and break necrosis by the underlying pathology i.e, adjacent to the
down of the blood brain barrier associated with reactive abscess wall, carcinoma, infarct, contusion or in the
astrogliosis. To address this hypothesis, immunohisto- mesial temporal lobes of the AD specimens. All raw values
chemical techniques were used to quantify reactive as- were reported as a mean6standard error of the means
B . Salhia et al. Brain Research 883 2000 87 –97
89
S.E.M. and also represented as a percentage of the endogenous GFAP was performed by incubating with a
maximum value obtained. The extent of astrocytosis was rabbit anti-GFAP antibody 1:10, Dako or mouse mono-
assessed by averaging the number of GFAP-positive clonal anti-VEGF antibody 1:10, UBI for 1 h at room
reactive astrocytes 4–6 high-powered fields HPF. MVD temperature. After three washes in PBS containing 1
counts were derived by averaging the number of Factor BSA and 0.9 NaCl, the sections were incubated with
VIII-positive vessels 4–6 HPF and VEGF staining was either Cy5-conjugated goat anti-mouse IgG 1:50, Jackson
quantified by averaging the percentage VEGF immuno- ImmunoResearch Laboratories Inc. or Cy3-conjugated
reactivity 4–6 HPF. goat anti-rabbit IgG 1:20, Jackson ImmunoResearch
In three cases of brain abscess and two cases of Laboratories Inc. for 1 h at room temperature. Sections
metastatic carcinoma, sections were stained for Ki-67 were washed in PBS and mounted for immunofluorescent
MIB-1, 1:50, Immunotech after microwave antigen re- microscopy. Sections were evaluated with the green
trieval and the proliferative index of the endothelium was immunofluorescence filter to detect expression of the GFP
obtained. The proliferative index was determined by transgene under the GFAP promoter and for either endog-
counting 1000 endothelial cell nuclei and determining the enous GFAP Cy3 or VEGF Cy5.
percentage staining positive for Ki-67. 2.2. Wild type and transgenic GFAP:GFP mouse
3. Results
