2 M
traumatic injury [5], ischemia [21] and in human Alzheim- numerous multinucleated cells, high mitotic activity, vas-
er’s disease [26]. Moreover, HO-1 expression in infiltrating cular proliferation and areas of focal necrosis. Seven
macrophages has been associated with disease severity in patients with oligodendroglioma received radiotherapy
atherosclerosis [33] and constitutes a marker of oxidative following the resection of the primary tumor, and 14
stress in asthma [8]. In brain tumors, elevated HO-1 patients received no post surgical treatment. In five pa-
expression was observed, but spatial and cellular expres- tients, no postsurgical therapy was mentioned thus sug-
sion patterns remain unresolved [6,22]. gesting that no post surgical therapy was applied. Thirteen
In order to provide a pathological basis for the in- patients
with anaplastic
oligodendroglioma received
volvement of HO-1 in oligodendrogliomas, we have radiotherapy after resection of the primary tumor, three
analyzed its expression in 69 oligodendroglioma tissue radiotherapy and chemotherapy with ACNU and VM26,
samples, in rat intracranially transplanted C6 gliomas, four and three received no postsurgical treatment. Involved-
rat brains and four neuropathologically unaltered human field radiotherapy was applied at doses of 36–60 Gy.
brains by immunohistochemistry. Twenty-six primary WHO grade II oligodendrogliomas and 16 primary WHO
2.2. Human control brains grade III anaplastic oligodendrogliomas were included.
Nineteen grade II tumors progressed, 10 were again grade Four control brains were obtained from autopsies at the
II oligodendrogliomas, and nine had progressed to higher Institute of Brain Research in Tuebingen Table 2. All
grade lesions. Eight anaplastic oligodendrogliomas pro- tissues were fixed in buffered 4 formalin pH 7.4 and
gressed, five were again WHO grade III tumors, and three embedded in paraffin by routine methods.
had progressed to glioblastoma multiforme. Double label- ing experiments confirmed the nature of HO-1 expressing
2.3. Cell culture cells. Reverse transcription–polymerase chain reaction
RT–PCR was used to demonstrate HO-1 mRNA. Rat C6 glioblastoma cell lines were obtained from the
American Type Culture Collection ATCC, Manassas, USA and raised in RPMI 1640 medium with Glutamax II
2. Materials and methods Gibco BRL, Paisley, UK containing 10 fetal calf serum
FCS, Gibco BRL, Paisley, UK and 1.2 penicillin 2.1. Oligodendroglioma patients
streptomycin Fluka, Buchs, Switzerland at 378C and 5 CO .
2
All oligodendrogliomas were resected at the Department ¨
of Neurosurgery in Tubingen or at the Department of 2.4. Intracranial transplantation and rat control brains
Neurosurgery of the Asklepios Klinik Schildautal in Seesen Table 1. Resection was documented by the
At near confluency, C6 glioblastoma cells were har- surgeons as incomplete or macroscopically complete. We
vested using a cell scraper and 5 ml were injected into the studied 42 oligodendroglioma tissue samples, 26 primary
basal ganglia region of male Sprague–Dawley rats at a
5
WHO World Health Organisation grade II oligodendro- concentration of 4310 ml [27]. After 2 weeks, rats were
gliomas and 16 primary WHO grade III anaplastic oligo- sacrificed, perfused with 4 paraformaldehyde and brains
dendrogliomas. Nineteen grade II tumors progressed, 10 were prepared as described. Control brains of healthy rats
were again grade II oligodendrogliomas and nine had were prepared as described above Table 2.
progressed to higher grade lesions. Eight anaplastic oligo- dendrogliomas progressed, five were again WHO grade III
2.5. Single labeling immunohistochemistry tumors, and three had progressed to glioblastoma mul-
tiforme. Histological diagnosis was performed by routine Five micrometer sections were deparaffinized and rehy-
neuropathology according to the WHO classification sys- drated. For antigen retrieval, the sections were immersed in
tem [9]. WHO grade II oligodendrogliomas were character- 0.01 M citrate buffer and irradiated in a microwave oven at
ized by enlarged rounded cells with a well-defined cell 750 W, five cycles of 5 min. Endogenous peroxidase was
membrane and clear cytoplasm around a central spherical blocked with 1 H O in methanol and the slices were
2 2
nucleus, uniformly round nuclei, high chromatin density, consequently incubated with nonspecific porcine serum.
low mitotic activity, microcalcifications and branching Rabbit polyclonal antibodies directed against recombinant
capillary network. WHO grade III oligodendrogliomas rat HO-1 StressGen, Victoria, Canada were diluted 1:200
were characterized by nuclear atypia, mitotic activity, high in 1 BSA bovine serum albumin TBS Tris-balanced
proliferation rate, rounded hyperchromatic nuclei, perinu- salt solution, pH 7.5, containing 0.025 M Tris, 0.15 M
clear swelling, few cellular processes and again mi- NaCl. Secondary antibody biotinylated anti-rabbit IgG
crocalcifications and branching capillary network. WHO Dako, Hamburg, Germany was diluted at 1:400 in BSA
grade IV glioblastoma multiforme were characterized by a TBS and applied to the slices for 30 min. Streptavidin–
high degree of cellular and nuclear polymorphism with biotin horseradish peroxidase complex Dako, Hamburg,
M .H. Deininger et al. Brain Research 882 2000 1 –8
3 Table 1
a
Oligodendroglioma patients and HO-1 immunoreactivity Primary tumors
Relapses Patient gender
Age Dig
HO-1 Rad
Chem Dig
HO-1 Surv ttp
Local Res
1 M 47
Ol 0.8
– –
36 LF
I 2 F
47 Ol
1.2 –
– 1
LF C
3 M 48
Ol 1.9
1 –
160 LFL
N A 4 M
53 Ol
0.5 N A
N A 164
RP N A
5 M 65
Ol 1.1
– –
0.1 RF
C 6 M
36 Ol
1.3 –
– 168
ROB I
7 F 53
Ol 1.4
1 –
77 RF
N A 8 M
50 Ol
0.8 –
– Ol
1.2 12
LFM N A
9 M 27
Ol 0.6
1 –
GB 33.6
96 LT
N A 10 F
36 Ol
1.3 –
– N A
N A 74
LFMB N A
11 F 32
Ol 1.2
N A N A
Ol 2.4
11 LTO
C 12 F
59 Ol
1.1 –
– Ol
4.3 59
RFP N A
13 M 46
Ol 0.8
N A N A
Ol 1.5
100 LP
N A 14 F
47 Ol
0.3 –
– Ol
16.4 22
RFP N A
15 M 46
Ol N A
1 –
AO 12.3
142 RFP
N A 16 M
39 Ol
0.8 1
– Ol
3.8 27
RT N A
17 F 41
Ol N A
– –
Ol 16.4
84 LTPO
I 18 M
34 Ol
1.1 –
– AO
13.7 43
LTM N A
19 M 49
Ol 0.4
– –
AO 2.4
22 LT
I 20 M
56 Ol
1.3 N A
N A AO
3.1 14
LTP N A
21 F 41
Ol 6.3
N A N A
Ol 4.7
52 RF
N A 22 M
22 Ol
N A 1
– Ol
1.3 78
LFB N A
23 F 35
Ol 0.4
– –
AO 4.1
36 LFB
I 24 M
45 Ol
0.8 –
– AO
3.8 42
RFP N A
25 M 51
Ol 0.7
1 –
AO 43.2
8 LFM
I 26 M
54 Ol
0.5 –
– Ol
17.6 108
LFL N A
27 F 59
AO 1.2
1 –
18 RTB
I 28 F
46 AO
9.3 1
1 10
LFP N A
29 M 78
AO 0.5
– –
2 LTB
I 30 M
53 AO
0.8 1
– 22
LP N A
31 F 26
AO 1.2
1 –
120 RFP
N A 32 F
55 AO
0.9 1
– 10
LP N A
33 F 70
AO 1.2
– –
1 RTM
I 34 F
43 AO
1.1 1
1 10
RFT I
35 F 47
AO 0.9
1 –
AO 15.9
28 LP
N A 36 F
33 AO
2.3 1
– GB
18.3 6
LF N A
37 M 52
AO 1.2
1 –
AO 41.2
42 RTP
N A 38 F
43 AO
2.2 1
– AO
12.4 65
RTPO N A
39 M 55
AO N A
– –
AO 4.5
12 RTPO
C 40 F
51 AO
1.8 1
1 GB
3.9 12
LPO I
41 F 48
AO 1.6
1 –
AO 1.8
24 RF
N A 42 M
39 AO
2.2 1
– GB
12.2 2
RF N A
a
Labeled cells are indicated as per cent of all counterstained nuclei. AO, anaplastic oligodendroglioma; B, basal; C, complete; Chem, chemotherapy; Dig, diagnosis; F, frontal; GB, glioblastoma multiforme; I, incomplete; L, left; Local, localization; M, medial; N A, not available; O, occipital; Ol,
oligodendroglioma; P, parietal; R, right; Rad, Radiotherapy; Res, resection; Surv, survival months; T, temporal; ttp, time to progression months.
Germany diluted 1:400 was subsequently applied for 30 Oxford, UK to rat tissues and GFAP, CD68 and HLA-DR,
min. Labeled antigen was visualized with standard -DP, -DQ macrophages, Dako, Denmark to human slices.
diaminobenzidine techniques Sigma, Deisenhofen, Ger- Visualization was achieved by adding rabbit anti-mouse
many. All sections were counterstained with hematoxylin. IgG diluted at 1:20 in TBS for 30 min and then APAAP
complex at a dilution of 1:100 in TBS for 30 min. 2.6. Double labeling experiments
Consecutively, we developed with Fast Blue BB salt Fluka, Buchs, Switzerland yielding a blue reaction
Slices were pretreated as described above. Then the product. To avoid antibody crossreactivity in double
differentiating mouse monoclonal antibodies were added to labeling experiments, slices were once more irradiated in a
the slices all at a dilution of 1:100 in TBS BSA. We added microwave for 20 min in citrate buffer [12]. Complete
anti-GFAP glial fibrillary acidic protein Boehringer, inhibition of alkaline phosphatase function was achieved as
Mannheim, Germany and ED1 macrophages, Serotec, previously described [2]. Consecutively, HO-1 was im-
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
