4 M
Table 2
a
HO-1 immunoreactivity in rat C6 gliomas and in rat and human control brains ID
Species Sex Age Histopathological diagnosis
HO-1 1
Rat M 6 weeks C6 glioma
43.8 2
Rat M 6 weeks C6 glioma
73.1 3
Rat M 6 weeks C6 glioma
45.6 4
Rat M 6 weeks C6 glioma
42.5 5
Rat M 6 weeks C6 glioma
62.9 6
Rat M 6 weeks Normal brain
7 Rat M 6 weeks
Normal brain 8
Rat M 6 weeks Normal brain
0.1 9
Rat M 6 weeks Normal brain
10 Rat M 6 weeks
Normal brain 0.4
11 Human M 39 years
Normal brain 12
Human M 26 years Normal brain
0.3 13
Human M 32 years Normal brain
0.2 14
Human F 39 years Normal brain
a
Labeled cells are indicated as per cent of all counterstained nuclei. F, female; M, male.
munolabeled as described above. No counterstain was mg mg tissue. cDNA was prepared using random hexa-
applied on double-labeled slices. mers, RNAsin
ribonuclease inhibitor and Moloney Mu- rine Leukemia Virus Reverse Transcriptase Promega,
Madison, WI. Samples were incubated 30 min at 378C and 2.7. Evaluation
5 min at 958C. Polymerase chain reaction was then performed using Thermus aquaticus Taq DNA Poly-
Positive cells were counted in five regions of solid merase Mini Kit Promega according to the manufactur-
tumor growth in WHO II and WHO III oligodendro- er’s instructions. The forward and reverse primers for
gliomas at 4003 magnification and compared to the total HO-1 were: 59-TGCCCAGCTCCTGGCCCGCCGCTT-39
number of counterstained nuclei in that area. In WHO IV and 59-GTGCATCAACACAGGCGCCTCTTC-39, respec-
glioblastomas and C6 gliomas of the rat, five regions in the tively, and gave a single band corresponding to a fragment
immediate vicinity of focal necrosis were evaluated to in human HO-1 cDNA [24]. RNA integrity of the ex-
demonstrate focal accumulation of HO-1 expressing cells. amined samples was confirmed using b-actin forward and
Then the percentage of HO-1 expressing cells was calcu- reverse primers: 59-TCACCCTGAAGTACCCCATCGAG
lated. Statistical analysis was performed using the Mann– -39 and 59-TTGGCCTTGGGGTTCAGGGGGG -39, re-
Whitney U-test. spectively, yielding a fragment in human beta-actin cDNA
[20]. Reaction conditions were: denaturation at 948C for 2 2.8. Controls and blocking experiments
min followed by 30 cycles of denaturation at 948C for 1 min, annealing at 608C, 558C HO-1, 558C b-actin,
Adjacent sections were stained omitting addition of elongation at 728C for 1 min. Finally, the reaction was
HO-1 antibody. HO-1 immunoreactivity was abolished incubated at 728C for 10 min. The resulting reaction
following overnight incubation of antibody with blocking products were analyzed on agarose gels stained with 0.5
peptide StressGen at 48C according to the manufacturer’s mg ml ethidium bromide.
instructions. No cross-reactivity is observed with HO-2. 2.9. RNA extraction and reverse transcriptase–
polymerase chain reaction RT–PCR
3. Results
Oligodendroglioma tissue of three primary WHO grade 3.1. Neuropathologically unaltered brains
II oligodendrogliomas, one WHO grade III anaplastic oligodendroglioma and four WHO grade IV glioblastoma
In rat and human control brains without neuropathologi- relapse patients were collected immediately after resection
cal alterations, HO-1 was expressed by singular astrocytes, and frozen in liquid nitrogen. Total RNA extraction was
neurons and macrophages situated in the cortex of the
performed using the RNeasy Mini Kit protocol as sug-
forebrain, diencephalon, cerebellum, and brainstem re- gested by the manufacturer Qiagen GmbH, Hilden, Ger-
gions. The number of HO-1 expressing cells was com- many. The RNA yields of all patients did not differ
parably low in rat Mean50.1, S.E.M.50.078 and human significantly and were typically in the range of 0.6–1.0
control brains Mean50.13, S.E.M.50.08 Table 2.
M .H. Deininger et al. Brain Research 882 2000 1 –8
5
3.2. Oligodendroglioma patients expressing macrophages were found in subendothelial
localizations of the vasculature. In human oligodendrogliomas Table 1, most promi-
nently, HO-1 expression was observed in macrophages 3.4. Double labeling experiments
microglial cells. In both, primary WHO grade II oligo- dendrogliomas Mean51.157, S.E.M.50.2477 and WHO
Double labeling experiments revealed the nature of HO- grade III anaplastic oligodendrogliomas Mean51.893,
1 expressing cells. In perinecrotic areas of rat C6 gliomas, S.E.M.50.5476, only singular HO-1 expressing macro-
the majority of HO-1 expressing macrophages microglial phages microglial cells were observed. However, signifi-
cells brown color coexpressed ED1 blue color Fig. cantly P50.0292 fewer HO-1 expressing cells were
1F. In contrary, the majority of HO-1 expressing cells did observed in primary WHO grade II oligodendrogliomas
not coexpress GFAP Fig. 1G. In human oligodendro- Fig. 1A than in primary anaplastic oligodendrogliomas.
glioma specimens, predominant colocalization of HO-1 Surprisingly, in areas of infiltrative oligodendroglioma
and CD68 Fig. 1H and HLA-DR, -DP, -DQ was ob- growth, higher numbers of HO-1 expressing cells were
served. Occasionally, double labeling of GFAP blue color observed than in areas of solid tumor growth. Here, HO-1
and HO-1 brown color was detected Fig. 1I. expressing cells were characterized by morphological
characteristics of neurons, astrocytes and macrophages 3.5. Blocking experiments
microglial cells. In oligodendroglioma Mean510.32, S.E.M.52.787, P,0.0001 and anaplastic oligodendro-
Antibody specificity was confirmed by blocking experi- glioma
relapses Mean513.78,
S.E.M.54.449, P5
ments with recombinant rat HO-1 peptide. The previously 0.0006, we observed significantly higher numbers of HO-
observed labeling pattern was completely abrogated data 1 expressing macrophages microglial cells than in the
not shown. primary tumors Fig. 1B. The most striking accumulation
of HO-1 expressing macrophages microglial cells was 3.6. Reverse transcriptase–polymerase chain reaction
observed adjacent to areas of focal necrosis in glioblas- RT–PCR
toma relapses Fig. 1C. Furthermore, in areas of necrosis, single disseminated macrophages microglial cells express-
HO-1 mRNA was not observed in WHO grade II ing HO-1 were readily detected. Prominent infiltration of
oligodendrogliomas lanes 1–3 Fig. 2, WHO grade III HO-1 expressing macrophages was frequently observed in
anaplastic oligodendroglioma lanes 4 or three WHO the walls of the tumor vasculature of high grade oligo-
grade IV glioblastoma lanes 6–8. In one glioblastoma dendroglioma relapses and glioblastoma multiforme re-
multiforme relapse lane 5, however, HO-1 mRNA was lapses.
detected. 3.3. Rat C6 gliomas
4. Discussion
