20 T
leaking vessels by immunofluorescence double staining Experiments were performed in male Fisher 344 rats
techniques [9]. Iffa Credo, France weighing 220–300 g. Animals were
Plasmin was shown to directly degrade ECM com- housed under standard conditions with free access to rat
ponents such as laminin and fibronectin [14,15]. Besides chow and tap water before and after surgery.
direct plasmin effects, activation of matrix metalloprotein- Irreversible occlusion of the right middle cerebral artery
ases MMPs by plasmin may play a crucial role in was performed as described previously [22,24]. Briefly,
ischemia-related ECM degradation. Pro-MMP 9 was animals were anesthetized with 2 isoflurane in a 70 30
shown to be converted to its active form MMP 9, v v nitrous oxide oxygen mixture and, using an oper-
gelatinase B in the presence of plasmin [17]. In accord- ating microscope Wild, the right MCA was exposed by a
ance with this study, pro-MMP 9 activation was reduced in subtemporal craniectomy. The artery and its lenticulo-
plasminogen deficient knock-out mice [13]. In a cell line striate branches were then occluded by bipolar electro-
experiment, pro-MMP 2 activation was inhibited by plas- coagulation. Afterwards, retracted soft tissues were re-
min inhibitors, suggesting a role of plasmin in the conver- placed, wounds were sutured, anesthesia was discontinued,
sion of pro-MMP 2 to its active form MMP 2, gelatinase and the rats were put back into their cages. Body tempera-
A as well [2]. ture was maintained at 378C by means of a rectal probe
The role of MMPs in focal cerebral ischemia has been connected to a heating pad CMA 150, Carnegie Medicine
addressed in several studies. In experimental focal cerebral during surgery and until animals regained consciousness.
ischemia and reperfusion in primates, MMP 2 was in- Thereafter, rectal temperature was checked frequently
creased after 1 h of middle cerebral artery occlusion every 10–15 min during the following 2 h and, if
MCAO and remained elevated for at least 7 days [10]. A necessary, it was corrected to 378C using a heating pad
similar increase was observed for MMP 9 in subjects with which was placed under the cage. After this, animals were
hemorrhagic transformation [10]. A clinical study in returned to their home cages and allowed free access to
humans revealed increased MMP 2 and 9 levels in the food and water.
cerebrospinal fluid of patients with acute stroke. These In order to verify successful MCAO and to visualize the
levels correlated with the extent of peri-infarct edema [3]. lesioned territory all brains were investigated by NMR
In a post-mortem study, MMP 2 and 9 were increased in imaging after 48 h of permanent ischemia. MRI inves-
infarcted brain regions 2 days and several months after tigations were performed on a Bruker DBX 47 30 4.7 T
stroke [5]. These studies suggest detrimental effects of equipped with an actively shielded gradient insert max.
MMP 2 and 9 in cerebral ischemia such as brain edema 100 mT m. RF pulses were transmitted and the MRI
1
and secondary hemorrhage. signal was acquired with a homogeneous birdcage
H Little is known about plasminogen activation in focal
resonator inner diameter, 3.5 cm. The lesion volume was cerebral ischemia. Investigations in mice showed increased
determined by means of T weighted quantitative in vivo
2
plasminogen activation in infarcted regions after 2 h of MRI [1]. Rats were anesthetized with 1–1.5 isoflurane
focal cerebral ischemia [27]. In previous experiments in delivered via a face mask and positioned with their heads
rats, plasminogen activation was increased in regions of in the resonator. Each animal was then subjected to one
structural damage after 3 h of focal ischemia and different imaging cycle, in which 13 continuous T
weighted
2
reperfusion intervals [19]. The time course of plasminogen coronal slices of the brain with a thickness of 1.2 mm were
activation in subacute stages days 2–7 of focal perma- taken using a RARE sequence optimized parameters:
nent cerebral ischemia has not yet been investigated. repetition time 3000 ms; effective echo time 66 ms; spatial
2
However, it may play a role in subacute hemorrhage and resolution in plane 156 mm . The total measuring time
edema formation after ischemic stroke. was 5 min.
To investigate plasminogen activation in long-term The animals survived for 48, 72, and 168 h groups 1, 2,
ischemia we used a rat model with permanent MCAO for 3; n54, 5, and 5. Thereafter, animals of group 2 and 3
2–7 days. animals of group 1 remained anesthetized after MRI were
anesthetized again and perfused with 50 ml ice-cold physiological saline, containing 1 bovine serum albumin,
2. Materials and methods 4 IE ml heparin, and 36 mg ml sodium nitroprusside at 5
ml min. The brains were removed and frozen in OCT 2.1. Animal model
embedding medium Sakura Finetec on dry ice and stored at 2808C until further processing.
All animal experiments were performed after ethical approval by the responsible government institution the
2.2. Preparation of cryostat sections ¨
‘Kantonales Veterinaramt’, Basel, Switzerland, reference number 1365. In accordance with these standards, every
Cryostat sections of 10 mm thickness were taken from effort was made to reduce the number of animals used and
regions 0–1 mm behind the bregma [18], were prepared at to ensure that they were free of pain and discomfort.
2208C, and stored at 2808C. To ensure comparability,
T . Pfefferkorn et al. Brain Research 882 2000 19 –25
21
adjacent sections were examined for MAP 2 immuno- acetone and chloroform, washed in phosphate buffer
histochemistry and zymographic plasminogen activation. solution PBS, and incubated with blotto 50 g non-fat
dry milk, 10 ml horse serum, 0.3 mmol l sodium azide 2.3. Zymographic detection of plasminogen activation
diluted in Tris–saline stock 38.5 mmol l Tris, 150 mmol l NaCl to reduce unspecific binding. After repeated
The method was described previously [21]. After thaw- washing in PBS, the sections were incubated with the
ing and drying at room temperature, sections were incu- secondary biotinylated antibody horse, anti-mouse, Vector
bated with an overlay consisting of commercial non-fat Laboratories, dilution 1:200, incubation period 2 h at
milk powder containing the plasmin substrate casein, 378C. This was followed by blocking endogenous per-
agarose, and human plasminogen. The overlay was pre- oxidase with H O for 20 min. Thereafter sections were
2 2
pared in several steps. Three different solutions were incubated with the ABC-Complex Vectastain-Elite-Kit,
prepared. Solutions 1 100 ml PBS124.6 mg MgSO 1 Vector Laboratories, incubation 30 min at 378C. Per-
4
13.2 mg CaCl and 2 10 ml dH O11.6 g commercial oxidase signal was developed with AEC AEC-Kit,
2 2
non-fat milk powder were separately heated to 508C, Biomeda. Nuclei were counterstained with Mayer’s hema-
solution 3 10 ml dH O1250 mg agarose to 708C. The toxylin Sigma, and tissue was blued in saturated sodium
2
solutions were mixed 0.75 ml of solution 1, 0.70 ml of bicarbonate.
solution 2, 0.50 ml of solution 3, and the mixture cooled to 378C. Then 100 ml of plasminogen 1.5 mg ml were
2.5. Morphometric analysis added. This mixture was evenly spread over the heated
sections 378C and immediately covered by a coverslip. To evaluate areas of visible plasminogen activation and
After incubation at 378C for 22 h the sections were decreased MAP 2 antigenicity, a morphometric program
immediately inspected for zones of overlay lysis and Optimas 6.5, Media Cybernetics, MD was used. Areas of
digitally scanned for later computer analysis. The incuba- plasmin-mediated lysis and MAP 2 loss were measured
tion period of 22 h was chosen, since plasmin-mediated and expressed as percent of the total ischemic hemisphere.
lysis had then reached a maximum. Longer incubation Affected areas were also evaluated separately for the basal
periods up to 36 h showed no further expansion of lytic ganglia and the cortex Fig. 1. To minimize the error of
regions. To exclude unspecific overlay lysis, control spatial resolution, adjacent brain sections were evaluated
sections of all groups were incubated with an overlay for zymography and MAP 2 immunohistochemistry. Cor-
lacking plasminogen. responding MRI images were selected from the individual
MRI series to illustrate overlapping of MRI changes with 2.4. Immunohistochemistry
overlay lysis and MAP 2 loss. This selection was guided by a rat brain atlas [18].
For MAP 2 detection, a monoclonal mouse antibody Boehringer Mannheim, dilution 1:800, incubation for 2 h
2.6. Statistical analysis at 378C, followed by 24 h at 48C was used. Before
incubation, sections were fixated in a 1:1 mixture of For statistical evaluation, data were presented as means
Fig. 1. A Drawing of a coronary section 10.70 mm bregma of the rat brain, adapted from Paxinos and Watson [18]. Areas of basal ganglia 3 and cortex 1 are marked, representing reference areas for quantification of plasminogen activation and MAP 2 loss. B Brain section of an animal with
long-term ischemia I5168 h using an overlay lacking plasminogen. No lysis is seen after 22 h of incubation.
22 T
and standard errors of mean S.E.M.. To analyse the time minogen, limited areas of ‘background’ overlay lysis were
course of overlay lysis and MAP 2 loss non-parametric seen in the non-ischemic control hemisphere. This lysis
testing Kruskal–Wallis, Mann–Whitney was performed. uniformly occurred in two different patterns: first, small,
To compare areas of overlay lysis with areas of MAP 2 pin-point-shaped regions with no preference for distinct
loss in individual sections the Wilcoxon matched-pairs anatomical structures; secondly, a circular rim of lysis
signed-ranks test was used. following pial structures around the section Fig. 2. In all
individual animals, this ‘background’ lysis affected less than 20 of the cortex and less than 10 of the basal
3. Results ganglia of the control hemisphere.
