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
stereotactic needle brain injury 3.1. Reactive astrocytosis, VEGF immunoreactivity and
To determine the temporal association between reactive neoangiogenesis in human CNS diseases
astrocytosis, neoangiogenesis and VEGF expression, a reproducible stereotactic needle stick injury model to the
The mean age of patients with the different diseases frontal cerebral cortex of mice was used [42,47,50]. A total
were 79 years — AD; 38 years — Abscess; 65 years — of 27 CD1 mice were anesthetized with 0.5 ml of 2.5
Peri-metastatic carcinoma brain; 52 years — Head Injury; Avertin 2,2,2-tribromoethanol and 2-methyl-2-butanol
71 years — Cerebral infarcts. Reactive astrocytosis with Sigma-Aldrich Chemical Company Inc., WI by intra-
increased numbers of large and darkly stained GFAP- peritoneal i.p. injection. The head was stabilized in a
positive reactive astrocytes were a prominent feature of all stereotactic frame and a burr hole drilled 2 mm anterior to
these disease processes compared to normal brain, al- the coronal suture and 2 mm lateral to the saggital suture.
though the degree of reactive astrocytosis varied with the The needle of a 5-ml Hamilton syringe was then inserted
type of pathology, as demonstrated in Table 1, Fig. 1. The through the burr hole to a depth of 2.5 mm for a duration
most exuberant reactive astrocytosis was induced in the of 1 min, then withdrawn. The contra-lateral frontal lobe
brain surrounding bacterial abscesses Table 1, Fig. 1-Row was used as the non-injured control side in all evaluations.
A, with an average raw value of 82628 GFAP-positive The track was made in cortical gray matter, not penetrating
astrocytes HPF. In comparison, diffuse head injury Fig. sub-cortical areas that normally contain higher numbers of
1-Row C, induced the lowest amount of reactive as- GFAP-positive cells [8]. All mice tolerated the procedure
trocytosis, with a raw value 2067 GFAP-positive as- well. Three mice were sacrificed on each of days 1–9
trocytes HPF, though this result should be tempered due to post-injury by cervical dislocation and the brains fixed in
our lack of knowledge of severity or the time of the formalin for 24 h and embedded in paraffin for histological
autopsy after the head injury. The amount of VEGF processing. Axial, 6-mm thick paraffin sections at right
immunoreactivity paralleled the degree of induced reactive angles to the needle tract were cut, stained with anti-GFAP,
astrocytosis, except for cerebral infarcts and peri-tumoral anti-Factor VIII and anti-VEGF antibodies and analyzed
brain, where VEGF immunoreactivity was disproportion- with the computer assisted image analysis system as
ately higher Table 1. The number of Factor VIII-positive above.
blood vessels MVD, as a measure of neoangiogenesis, Three FVB N background GFAP:GFP transgenic mice
was related to the degree of reactive astrocytosis and a gift from Dr. A. Messing, Madison, WI, USA under-
VEGF immunoreactivity. This is best exemplified by went a similar needle tract injury and were evaluated 7
MVD, raw counts of 2063 vessels HPF, associated with days after injury with immunofluorescence microscopy.
the florid reactive astrocytosis in bacterial abscess, com- Mice were sacrificed by cervical dislocation. Brains were
pared to only 661 vessels HPF in brains with prior head removed within 5 min of sacrifice, embedded in OCT
injury, AD or infarcts. In these latter conditions, the degree Tissue Tek embedding medium on dry ice and stored at
of reactive astrocytosis and associated neoangiogenesis 2708C until further processing. Axial cryostat sections
although relatively low compared to brain around bacterial were fixed in ice cold acetone for 3 min at 2208C, washed
abscess, was still significantly high relative to normal brain with PBS for 10 min and blocked in a solution of PBS
Table 1. In all cases, astrocytosis, VEGF immunoreactivi- containing 1 BSA and 0.9 NaCl for 30 min at room
ty and MVD was increased from normal brain Fig. 1-Row temperature. Double immunofluourescence for VEGF and
F. In addition, blood vessels associated with brain abscess
90 B
Table 1
a
Semi-quantitative analysis of immunohistochemical staining in a variety of human neuropathologies Diagnosis
n Reactive
VEGF Vascularity
Astrocytosis Expression
No. Factor No. GFAP-
VEGF VIII-stained
Positive immunoreactivity
vessels HPF Cells HPF
HPF Normal
1 261 2
461 9 261 10
Alzheimer’s disease 4
3962 48 2564 54
661 30 Bacterial abscess
3 82628 100
661 100 2063 100
Head injury 3
2067 24 1364 28
661 30 Infarct
6 2263 27
2363 50 661 30
Peritumoral brain 4
2861 34 3061 65
1163 55
a
Reactive astrocytosis, VEGF expression and vascularity in a spectrum of human neuropathological specimens. Raw mean values6S.E.M. are shown and values are reported as a percentage of the maximum in brackets. All slides were quantified at 4003magnification. VEGF, vascular endothelial growth
factor; S.E.M., standard error of the mean; GFAP, glial fibrillary acidic protein; HPF, High Power Field.
appeared larger and more robust than other CNS diseases parameters subsequently decreased towards normal levels
investigated and the blood vessels associated with head by day 9. While the peak days for each parameter analyzed
injury seem much smaller compared to other conditions. In do not occur on the same day in this experimental
the astrogliotic response associated with AD and cerebral paradigm, the overall temporal correlation between them is
infarcts, the vascularity, although increased from normal clearly demonstrated. The GFAP:GFP transgenic mice
brain, was relatively less compared to the degree of VEGF were used for co-localization experiments in a needle stick
immunoreactivity by reactive astrocytes. The proliferative injury model to confirm that the reactive astrocytes in-
index of endothelial cells ranged from 8 to 12 in brain duced by the needle tract injury in Fig. 4 were
abscesses n53 and from 2 to 6 n52 near metastatic expressing VEGF. Green immunofluorescence denoted the
tumor. These results clearly demonstrate active neoan- expression of the GFP transgene under regulation of the
giogenesis in these two conditions. Material taken from GFAP promoter Fig. 4A. These same GFP expressing
autopsy specimens was not used to determine proliferative cells were reactive astrocytes, as they also expressed
index because, in our experience, detection of this antigen endogenous GFAP as detected by double immunofluores-
is not reliable in tissue undergoing prolonged fixation. cence, where the cells are yellow due to the combination of
green and the Cy3 secondary antibody used to detect 3.2. Temporal and spatial expression of reactive
endogenous GFAP Fig. 4B. On adjacent sections, the astrocytosis
, VEGF immunoreactivity and GFP reactive astrocytes are the same cells that are express-
neoangiogenesis ing VEGF, where they appear red due to combination of
green and Cy5 immunofluorescence used to detect VEGF The mouse stereotactic needle stick injury resulted in a
Fig. 4C. There were no significant reactive GFP-positive distinct tract that could be consistently visualized on the
astrocytes in the non-injured contra-lateral hemisphere injured side with a surrounding reactive astrogliotic re-
Fig. 4D. sponse Fig. 2A. The injury induced GFAP-positive
astrocytes Fig. 2B to express VEGF Fig. 2C with accompanying neoangiogenesis, as marked by Factor VIII-
4. Discussion
