A rat model of focal embolic cerebral ischemia

Brain Research 766 Ž1997. 83–92

Research report

A rat model of focal embolic cerebral ischemia Rui Lan Zhang a , Michael Chopp a

a,b,)

, Zheng G. Zhang a , Quan Jiang a , James R. Ewing

a

Department of Neurology, Henry Ford Health Sciences Center, Detroit, MI 48202, USA b Department of Physics, Oakland UniÕersity, Rochester, MI 48309, USA Accepted 15 April 1997

Abstract We developed a new model of embolic cerebral ischemia in the rat which provides a reproducible and predictable infarct volume within the territory supplied by the middle cerebral artery ŽMCA.. The MCA was occluded by an embolus in Wistar rats Ž n s 71.. An additional three non-embolized rats were used as a control. Cerebral blood flow ŽCBF. was measured by means of laser Doppler flowmetry ŽLDF. and perfusion weighted imaging ŽPWI. before and after embolization. The evolution of the lesion was monitored by diffusion weighted imaging ŽDWI.. Cerebral vascular perfusion patterns were examined using laser scanning confocal microscopy. Infarct volumes were measured on hematoxylin and eosin ŽH & E. stained coronal sections. The lodgment of the clot at the origin of the MCA and the ischemic cell damage were examined using light microscopy. Regional CBF in the ipsilateral parietal cortex decreased to 43 " 4.1% Ž P - 0.05. of preischemic levels Ž n s 10.. Confocal microscopic examination revealed a reduction of cerebral plasma perfusion in the ipsilateral MCA territory Ž n s 6.. MRI measurements showed a reduction in CBF and a hyperintensity DWI encompassing the territory supplied by the MCA Ž n s 4.. An embolus was found in all rats at 24 h after embolization. The infarct volume as a percentage of the contralateral hemisphere was 32.5 " 3.31% at 24 h Ž n s 20., 33.0 " 3.6% at 48 h Ž n s 13., and 34.5 " 4.74% at 168 h Ž n s 12. after embolization. This model of embolic focal cerebral ischemia results in ischemic cell damage and provides a reproducible and predictable infarct volume. This model is relevant to thromboembolic stroke in humans and may be useful in documenting the safety and efficacy of fibrinolytic intervention and in investigating therapies complementary to antithrombotic therapy. q 1997 Elsevier Science B.V. Keywords: Embolus; Focal cerebral ischemia; Rat; MRI; Laser confocal microscopy

1. Introduction There are three primary stroke subtypes: thrombosis, embolism, and hemorrhage w1,16x. Thrombosis and embolism are responsible for approximately 80% of human stroke w1,16x. Models of focal cerebral ischemia induced by intraarterial autologous or heterologous blood clot embolization have provided much information on the safety and efficacy of antithrombotic therapies w6,10,12,14,22x. However, these models lack reproducibility and uniformity in the size and location of the infarcts, because placement and ultimate lodgment of the emboli are not controlled. Therefore, there is a need for an animal model with a

predictable localization and reduction of cerebral blood flow and a reproducible focal cerebral infarction to mimic the large cerebral artery thromboembolic stroke in the human. We have recently developed a rat model of thrombotic focal cerebral ischemia which exhibits a reproducible infarct volume w21x. In the present study, we describe a rat model of embolic focal cerebral ischemia, in which the middle cerebral artery ŽMCA. is selectively occluded by a fibrin-rich embolus. The lodgment of the clot, reduction in cerebral blood flow ŽCBF. and subsequent cerebral infarct volume were highly reproducible.

2. Materials and methods

)

Corresponding author. Henry Ford Hospital, Neurology Department, 2799 West Grand Blvd., Detroit, MI 48202, USA. Fax: q1 Ž313. 876-1318. 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 5 8 0 - 5

All experimental procedures have been approved by the Care of Experimental Animals Committee of Henry Ford Hospital.

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R.L. Zhang et al.r Brain Research 766 (1997) 83–92

2.1. General surgical preparation Male Wistar rats weighing 320–400 g Ž n s 74. were used in the experiments. Animals were anesthetized with 3.5% halothane, and anesthesia was maintained with 1.0% halothane in 70% N2 O and 30% O 2 using a face mask. Rectal temperature was maintained at 37 " 0.58C throughout the surgical procedure using a feedback regulated water heating system. The right femoral artery and vein were cannulated with a PE-50 catheter for continuous monitoring of blood pressure and measurement of blood gases ŽpH, pO 2 , pCO 2 . and for drug administration, respectively. 2.2. Preparation of the embolus The method used to prepare a white embolus was adapted and modified from Overgaard et al. w11x. Briefly, femoral arterial blood from a donor rat was withdrawn into 20 cm of PE-50 tubing and retained in the tube for 2 h to clot at room temperature, and subsequently retained for 22 h at 48C. Five cm of the PE-50 tube containing clot was cut and attached at each end to a 20 cm PE 10 tube interconnected by a syringe filled with saline. The clot was shifted by continuous alternating movement from one syringe to the other for 5 min. A single clot Ž25 mm = 3.14 = 0.01 mm2 s 0.8 m l. was transferred to a modified PE-50 catheter with a 0.3 mm outer diameter filled with saline.

Fig. 1. Schematic drawing of the catheter placed into the external carotid artery ŽECA. and the internal carotid artery ŽICA. of the rat, with its tip at 2 mm from the origin of the middle cerebral artery ŽMCA.. The catheter contains a clot Žblack..

2.3. Animal model Under the operating microscope ŽCarl Zeiss Inc., Thornwood, NY, USA. the right common carotid arteries ŽCCA., the right external carotid artery ŽECA. and the internal carotid artery ŽICA. were isolated via a midline incision. A 5-0 silk suture was loosely tied at the origin of the ECA and ligated at the distal end of the ECA. The right CCA and ICA were temporarily clamped using a curved microvascular clip ŽCodman & Shurtleff Inc., Randolf, MA, USA.. A modified PE-50 catheter with a 0.3 mm outer diameter filled with a 25 mm clot, which was attached to a 100-m l Hamilton syringe, was introduced into the ECA lumen through a small puncture. To evaluate the optimal length of a clot for consistent lodgment at the origin of the MCA, various lengths Ž10–30 mm. of a clot were tested in a preliminary study. The probability of lodgment of a clot at the origin of the MCA was 40% in rats injected with a clot less than 25 mm in length and was 95% with a 25 mm long clot. Twenty percent of animals injected with a clot longer than 25 mm exhibited a hemispheric infarction. A clot less than 25 mm in length frequently lodged within a branch of the MCA or the ACA. In contrast, a clot longer than 30 mm blocked the ACA, the MCA and the PCA. Therefore, in the present study, a 25 mm long clot was selected for intraluminal placement. The suture around the

origin of the ECA was tightened around the intraluminal catheter to prevent bleeding, and the microvascular clip was removed. A 15 mm length of catheter was gently advanced from the ECA into the lumen of the ICA. At this point, the intraluminal catheter was 2–3 mm from the origin of the MCA. The clot along with 5 m l of saline in the catheter was injected into the ICA over 10 s ŽFig. 1.. The catheter was withdrawn from the right ECA 5 min after injection. The right ECA was ligated. The duration of the entire surgical procedure was approximately 25 min. Heparin was not administered to any animal. To evaluate the effects of placement of the catheter and injection of saline on ischemic cell damage, three rats were subjected to the same surgical procedures but without intravascular insertion of an embolus. 2.4. Neurologic deficits Neurologic examinations were performed at 2 h, 24 h, 48 h and 168 h after injection of a clot. The neurologic findings were scored as described by Zea Longa et al. w20x with some modification on a 5-point scale: no neurologic deficit s 0; right Horner’s syndromes 1; failure to extend left forepaw fully s 2; turning to left s 3; and circling to left s 4.

R.L. Zhang et al.r Brain Research 766 (1997) 83–92

2.5. Blood analysis Arterial blood gas ŽpH, pCO 2 , and pO 2 . was measured before and 1 h after injection of a clot. 2.6. Monitoring of regional CBF by LDF With the rat mounted on a stereotactic frame, relative regional CBF ŽrCBF. was measured using laser Doppler flowmetry ŽLDF. w21x. LDF was performed with a PeriFlux PF4 flowmeter ŽPerimed AB, Jarfalla, ˚ ˚ Sweden. with relative flow values expressed as perfusion units. A 1.5 mm diameter burr hole was placed 1.0 mm posterior and 5.5 mm lateral to the bregma in each hemisphere w13x. The dura was left intact and mineral oil was placed onto the burr hole. Using a micromanipulator, two probes were positioned 0.5 mm above the dural surface, being careful to avoid any large vessels. Regional CBF in two hemispheres was simultaneously measured for 20 min prior to injection of a clot Žto calculate the baseline flow. and immediately after injection of a clot and continuously until 2 h after injection of a clot. In a previous study, we have demonstrated that changes in rCBF were not significant when probes were repositioned w21x. The data were continuously stored in a computer and analyzed using Perimed data acquisition and analysis system ŽPerimed AB, Jarfalla, ˚ ˚ Sweden.. Regional CBF was expressed as a percentage of preischemic baseline values. 2.7. MRI measurements Diffusion weighted imaging ŽDWI. and perfusion weighted imaging ŽPWI. were performed on animals subjected to embolic stroke. MRI measurements were performed using a 7 T, 20 cm bore superconducting magnet ŽMagnex Scientific, Abingdon, UK. interfaced to a console ŽSMIS, Surrey, UK.. A 12 cm bore gradient coil set, capable of producing magnetic field gradients up to 20 gaussrcm and a birdcage coil with 5 cm inter-diameter were used. Images were produced using a 3.2 cm field of view ŽFOV., 2 mm slice thickness and 128 = 64 image matrix. Stereotaxic ear bars were used to minimize movement during the imaging procedure. While in the magnet, animals were anesthetized using a face mask with a 70% of N2 O and 30% of O 2 gas mixture and halothane Ž0.75– 1%.. Rectal temperature was maintained at 378C using a feedback controlled water bath. A modified FLASH w4x imaging sequence was employed for reproducible positioning of the animal in the magnet at each DWI session. DWI was performed prior to injection of a clot, continuously for 5 h and at 24 h, and 48 h after embolization. PWI was performed using a technique described by Williams et al. w19x. Perfusion images were obtained prior to injection of a clot and at various times between 1–5 h and at 1 and 2 days after injection of a clot. Perfusion measurement MRI parameters were: TR s 1 s, TE s 30

85

ms, 64 = 64 image matrix, 2 mm slice thickness, and a 3.2 cm field of view. The duration of the inversion pulse was 1 s at a power level of 0.5 W. Relative CBF was calculated w19x. After the final MRI measurement, the brain was removed, and 6 m m thick coronal sections were obtained and stained with H & E for evaluation of ischemic cell damage. 2.8. Histopathology 2.8.1. Light microscopy Animals were anesthetized Ži.m.. with ketamine Ž44 mgrkg. and xylazine Ž13 mgrkg.. Rats were transcardially perfused with heparinized saline and 10% buffered formalin, and brains were removed. Using a rat brain matrix, each brain was cut into 2 mm thick coronal blocks, for a total of 7 blocks per animal. The brain tissue was processed, embedded, and 6 m m thick paraffin coronal sections from each block were cut and stained with H & E for histopathological evaluation. Gross hemorrhage, defined as blood evident to the unaided eye on the two faces of each block and on the H & E stained sections, was evaluated in each animal. The lodgment of a clot at the origin of the MCA was recorded before each brain was cut into coronal blocks. Phosphotungstic acid hematoxylin ŽPTAH. staining was performed on 6 m m thick coronal cerebral sections to examine fibrin deposition w5,7x. 2.8.2. Confocal microscopy Cerebral vascular perfusion patterns were examined using a double labeled plasma flow method. Evans blue Ž2% solution in saline, 0.2 mlr100 g body weight; Sigma Chemicals, St. Louis, MO. was injected intravenously 5 min prior to injection of a clot. One ml of fluorescein isothiocyanate ŽFITC.-dextran Ž50 mgrml, 2 = 10 6 molecular weight; Sigma, St. Louis, MO. was administered i.v. 2 h after injection of an embolus and allowed to circulate for 1 min. The animals Ž n s 6. were decapitated and the brains were rapidly removed and placed in 4% of paraformaldehyde for 24 h. Vibratome coronal sections Ž100 m m. were analyzed with a Bio-Rad MRC 1024 laser scanning confocal microscope mounted onto a Zeiss microscope ŽBio-Rad, Cambridge, MA.. A 10 = or 40 = objective was used for data acquisition. Areas of interest were scanned in 512 = 512 pixel format in the xry direction using a 4 = frame scan average. Twenty or 60 thin optical sections were scanned through a specimen along the z-axis with a 5 m m or 1 m m step size. 2.8.3. Measurement of infarct Õolume Volume of cerebral tissue infarction was measured using a Global Lab Image analysis program ŽData Translation, Marlboro, MA.. Each H & E stained coronal section was evaluated at 2.5 = magnification. The area of infarc-

R.L. Zhang et al.r Brain Research 766 (1997) 83–92

86 Table 1 Physiological parameters

Pre Ž ns 20. 1 h Ž ns 20. 4 h Ž ns8.

pH

pCO 2 ŽmmHg.

pO 2 ŽmmHg.

7.43"0.01 7.42"0.01 7.45"0.01

35.5"1.4 39.4"1.6 34.3"2.1

139"3 136"6 135"4

Values are mean"S.E. and obtained at pre-embolization 1 h and 4 h after injection of clot.

tion and the area of both hemispheres Žmm2 . were calculated on H & E stained sections by tracing the areas on the computer screen, and the volumes Žmm3 . were determined by integrating the appropriate area with the section interval thickness. To reduce errors associated with processing of tissue for histological analysis, the area of infarction in each section was presented as the percentage of the infarct to the area of the contralateral hemisphere, and the infarct volume was also presented as the percentage of infarct volume to the volume of the contralateral hemisphere Žindirect volume calculation. w8,17x.

Fig. 2. Regional cerebral blood flow measured using LDF in the parietal cortex prior to and 2 h after embolization Ž ns10.. ŽB. ipsilateral and ŽI. contralateral.

presented as means " S.E. Statistical significance was set at P - 0.05.

3. Results 3.1. Physiological parameters

2.8.4. Experimental protocols To evaluate embolus formation and the subsequent induction of a focal ischemic lesion, the following experiments were performed: Ž1. animals were sacrificed 24 h Ž n s 20., 48 h Ž n s 13. and 168 h Ž n s 12. after injection of a clot and the volume of cerebral infarction was measured; Ž2. CBF measurement to correlate relative rCBF reduction measured by LDF with cerebral infarct volume: animals Ž n s 10. were sacrificed 168 h after injection of a clot and infarct volume was measured; Ž3. confocal microscopy to examine cerebral vascular perfusion patterns after embolization: animals were sacrificed 1 h Ž n s 3. or 2 h Ž n s 3. after injection of a clot; Ž4. diffusion and perfusion MRI to monitor ischemic tissue after embolization Ž n s 4..

The arterial blood gas values were within a normal physiological range ŽTable 1.. The mean arterial blood pressures ŽMABP. were in a normal physiological range prior to injection of a clot Ž108 " 6 mmHg. and 1 h after injection of a clot Ž104 " 3 mmHg.. 3.2. Neurological deficits Animals exhibited moderate to severe neurological deficit 1 h after injection of a clot and mild to moderate at 24 h, 48 h and 168 h after embolization ŽTable 2.. 3.3. rCBF As measured by LDF, regional CBF in the ipsilateral parietal cortex decreased to 43 " 4.1% Ž P - 0.05. of preinjection levels immediately after embolization and was sustained during 2 h of measurement. rCBF in the contralateral parietal cortex was not significantly different from preischemic values ŽFig. 2.. The mean percent reduc-

2.9. Statistics Data were analyzed using a paired t-test in changes of rCBF and a linear regression analysis in correlation of reduction in rCBF with infarct volume. All values are

Table 2 Neurological deficit Number of rats exhibiting neurological scores 1h

24 h

48 h

168 h

Scores

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

24 h Ž n s 20. 48 h Ž n s 13. 168 h Ž n s 12.

0 0 0

0 0 0

16 3 3

1 9 7

3 1 2

0 0 0

16 3 3

2 8 6

2 2 3

0 0 0

– 0 0

– 4 3

– 8 8

– 1 1

– 0 0

– – 0

– – 4

– – 8

– – 0

– – 0

R.L. Zhang et al.r Brain Research 766 (1997) 83–92

87

Table 3 The distribution of lodgment of a clot

Fig. 3. ŽA. Hematoxylin and eosin-stained coronal section from a representative rat obtained 24 h after injection of a clot. An embolus occluded the right intracranial segment of the ICA at the origin of the MCA Žarrow.. The left intracranial segment of ICA was patent. The right hemisphere exhibited an infarct in the territory of the MCA. ŽB. Phosphotungstic acid hematoxylin ŽPTAH. staining of coronal section of a rat 24 h after embolization shows fibrin strands in the embolus occluding the intracranial segment of the ICA at the origin of the MCA.

R.L. Zhang et al.r Brain Research 766 (1997) 83–92

88

tion of cerebral blood flow over the 2 h post-embolization period was highly correlated with the final Ž168 h. percent volume of cerebral infarction Ž r s 0.84, p s 0.003.. 3.4. Histopathology-light microscopy Two rats from the 48 h Ž n s 1. and 168 h Ž n s 1. groups died at 24 h and 72 h after embolization. The autopsy showed massive cerebral edema. Subarachnoid hemorrhage was detected in four rats Ž1 in the 24 h group, 2 in the 48 h group and 1 in the 168 h group. at the time of sacrifice. These rats Ž n s 6. were excluded from data analysis. Injection of a clot resulted in embolization at the origin of the MCA ŽFig. 3A.. An embolus was present in 100% Ž20 of 20. at 24 h, 62% Ž8 of 13. at 48 h, and 25% Ž3 of 12. at 168 h after injection of a clot, respectively. The intraarterial location of the clots is depicted in Table 3. Fibrin deposition in the embolus was confirmed by PTAH staining ŽFig. 3B.. Gross hemorrhage was located in the ipsilateral subcortex and was detected in 10% Ž2 of 20., 15% Ž2 of 13., and 8% Ž1 of 12. of rats at 24 h, 48 h and 168 h after embolization, respectively. The infarct volume in rats was 32.5 " 3.31% at 24 h Ž n s 20., 33.0 " 3.6% at 48 h Ž n s 13., and 34.5 " 4.74% at 168 h Ž n s 12.. Table 4 summarizes the absolute values of the contralateral and the ipsilateral hemispheric volumes, indirect infarct volumes and the percent infarct volumes to the contralateral hemisphere. Eosinophilic neurons Žred neurons. and neurons with a loss of cellular integrity and affinity for hematoxylin staining Žghost neurons. were evident in the ischemic lesion at 24 h and 48 h after embolization ŽFig. 4A and B.. Macrophages were prominent in the ischemic tissue at 168 h ŽFig. 4C.. The ischemic neuronal damage was not detected in control rats Ž n s 3.. 3.5. Confocal microscopy Cerebral microvessels perfused only prior to the embolization exhibited red fluorescence ŽEvans blue stain, Fig. 5C and G., while those only perfused before sacrifice showed green fluorescence ŽFITC-dextran, Fig. 5D and H.. Microvessels filled with both Evans blue and FITC-dextran displayed yellow fluorescence ŽFig. 5B and F.. Red and yellow fluorescence were present in the lumen of the Table 4 The absolute hemisphere volumes and indirect infarct volumes Hemisphere Žmm3 .

24 h Ž ns 20. 48 h Ž ns13. 168 h Ž ns12.

Contralateral

Ipsilateral

Infarct Žmm3 .

662.14"16.7 633.47"20.4 667.15"16.1

667.0"15.3 655.67"22.7 655.67"25.8

213.05"21.7 204.84"20.7 227.09"29.4

Values are mean"S.E.

Fig. 4. Photomicrographs of H&E staining coronal sections obtained from representative rats after 24 h ŽA., 48 h ŽB. and 168 h of embolization. Red neurons ŽA, arrows., ghost neurons ŽB, arrows. and macrophages were detected.

ipsilateral cerebral microvasculature in the MCA territory ŽFig. 5A and B. and only yellow fluorescence was visualized in the contralateral hemisphere ŽFig. 5E and F.. Unilateral plasma perfusion deficits Žred fluorescence. were detected at 1 h Ž n s 3. and 2 h Ž n s 3. after embolization. 3.6. MRI Serial MRI perfusion measurements revealed a reduction in CBF encompassing the territory supplied by the

R.L. Zhang et al.r Brain Research 766 (1997) 83–92 Fig. 5. Confocal microscopy of a vibratome coronal section Ž100 m m. of a rat brain 2 h after injection of a clot. ŽA. to ŽD. are from a homologous area within the ipsilateral cortex and ŽE. to ŽH. are from a homologous area within the contralateral cortex. ŽA. and ŽE. are low magnification Ž10=, 100 m m.. ŽB. to ŽD. and ŽF. to ŽH. are high magnification images Ž40=, 60 m m.. A double fluorescein plasma method shows red fluorescence ŽEvans blue; C and G. prior to embolization and green fluorescence ŽFITC-dextran; D and H. 2 h after embolization. An absence of perfused microvessels Žred fluorescence. resulting from the placement of the embolus is noted in the ipsilateral cortex ŽA and B.. The yellow fluorescence indicates vessels perfused after embolization. All microvessels Žyellow fluorescence. were perfused in the contralateral cortex ŽE and F..

89

90 R.L. Zhang et al.r Brain Research 766 (1997) 83–92 Fig. 6. ŽA. Perfusion-weighted images of coronal sections from a representative rat brain obtained prior to and at 1 h to 48 h after embolization. A rapid decline in signal intensity indicating a reduction of CBF was observed in the MCA territory. Signal intensity remained low at 24 h after injection of a clot. Signal intensity returned in the cortex at 48 h after injection of a clot. ŽB. Diffusion-weighted images of coronal sections from the same animal brain in ŽA. showing the evolution of the diffusion weighted hyperintensity. An H&E stained coronal section shows the infarct area in the same rat.

R.L. Zhang et al.r Brain Research 766 (1997) 83–92

right MCA ŽFig. 6A.. CBF values in the ipsilateral hemisphere after injection of a clot declined from the preinjection CBF values and remained low at 4 h and 24 h after embolization. However, the CBF in the right cortex increased towards preinjection values at 48 h after embolization. CBF values in subcortex remained low 48 h after injection of a clot. The contralateral hemisphere exhibited a small reduction in CBF after injection of a clot. Fig. 6B presents DWI from the rats showing a reduction of CBF in Fig. 6A obtained at 60 min, 4 h, 24 h and 48 h after embolization. Hyperintensity was apparent in the DWI of the ipsilateral MCA region at 60 min after injection of a clot; image contrast incorporating T2 weighting tended to increase as time progressed and persisted up to 48 h after embolization. Hyperintense regions in the DWI corresponded to the infarct area observed in H & E sections ŽFig. 6C.. Similar profiles of MRI perfusion and DWI changes were detected in all the animals Ž n s 4.. 4. Discussion In the present study, we have presented a new model of embolic focal cerebral ischemia in the rat. The major advantage of this model over other embolic models is that a fibrin-rich embolus is induced at the origin of the MCA, and this model provides a reproducible and predictable infarct volume within the territory supplied by the MCA. The embolic focal cerebral ischemia models in rats and rabbits previously reported are induced by injection of a clot or clots through the extracranial segment of the ICA w6,11x. Although there is the high probability that the MCA is the recipient site for emboli, uniformity in the size and location of the infarcts in these models is not achieved due to inherent technical difficulties involved in the uncontrolled placement and ultimate lodgment of the multiple emboli. Location of the MCA occlusion determines infarct size w15,18x. We overcome this limitation by locally delivering an intact fibrin-rich embolus into the segment of the intracranial ICA 2–3 mm proximal to the origin of the MCA. An embolus was detected in all rats sacrificed at 24 h after embolization and 98% of all injected emboli were lodged at the origin of the MCA. After clot injection, rCBF was decreased to 43% of preembolization levels in the ipsilateral MCA territory and this decreased rCBF persisted for at least 2 h after embolization. The percentage reduction of rCBF is comparable to the values obtained from a thrombotic model of MCA occlusion w21x. A significant decrease of rCBF is accompanied by a corresponding reduction of plasma perfusion in the ipsilateral MCA territory. In the present study, using a double fluorescein plasma method we have demonstrated a reduction of cerebral plasma perfusion after embolization. In addition, there was a high correlation between the reduction of rCBF measured with LDF and infarct volume.

91

The neurological deficit scores are in agreement with values obtained from the suture and thrombotic models, in which animals exhibited moderate to severe neurological deficit at acutely and a mild to moderate neurological deficit chronically after MCA occlusion w5,7,21x. Infarct volume in this model is consistent with that obtained in the suture model of MCA occlusion and thrombotic model of focal ischemia in rats w2,21x. The ischemic neuronal damage and infarction at 24 h to 168 h after embolization are confirmed on the H & E sections. Since the fibrin-rich embolus blocks an intracranial segment of the ICA at the origin of the MCA, a relatively uniform predictable infarct volume is produced. This is an important end point if therapeutic interventions are to be investigated. Gross hemorrhagic infarct was observed in 11% of animals. Autopsy studies reveal that 18–42% of infarcts are grossly hemorrhagic in patients with ischemic stroke w3x. Gross hemorrhage occurs most often in embolic stroke in the human. The lower rate of hemorrhage in the animal model may be related to the utilization of young rats. The evolution of the embolic lesion in this model can be monitored by measuring diffusion and perfusion weighted MRI. The hyperintensity observed on DWI after embolization is consistent with data obtained from the suture model of MCA occlusion and thrombotic model of focal ischemia in rats w9,21x. In summary, we have demonstrated that a rat model of embolic focal cerebral ischemia induced by a fibrin-rich clot results in ischemic neuronal damage and provides a reproducible and predictable infarct volume. This model of embolic ischemia is relevant to thromboembolic stroke in humans and may be useful in determining the safety and efficacy of fibrinolytic interventions and in investigating of therapies complementary to antithrombotic therapy. Acknowledgements The authors wish to thank Cecylia Powers and Xiuli Zhang for technical assistance, Dr. Anton Goussev and Qin Wang for data analysis and Denice Janus for manuscript preparation. This work is supported in part by NINDS Grants PO1 NS23393, RO1 NS33627 and RO1 NS34184. References w1x G.W. Albers, Antithrombotic agents in cerebral ischemia, Am. J. Cardiol. 75 Ž1995. 348–388. w2x M. Chopp, R.L. Zhang, H. Chen, Y. Li, N. Jiang, J.R. Rusche, Post ischemic administration of anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat, Stroke 25 Ž1994. 869–876. w3x C.M. Fisher, R.D. Adams, Observation on brain embolism with special reference to hemorrhagic infarction, in: A.J. Furlan ŽEd.., The Heart and Stroke, Springer-Verlag, BerlinrHeidelberg, 1987, pp. 16–36.

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w4x J. Frahm, A. Hasse, D. Matthaei, Rapid NMR imaging of dynamic processes using the FLASH technique, Magn. Res. Med. 3 Ž1986. 321–327. w5x N. Futrell, C. Millikan, B.D. Watson, W.D. Dietrich, M.D. Ginsberg, Embolic stroke from a carotid arterial source in the rat: pathology and clinical implications, Neurology 39 Ž1989. 1050– 1056. w6x M.G. Hamilton, J.S. Lee, P.J. Cummings, J.M. Zabramski, A comparison of intra-arterial and intravenous tissue-type plasminogen activator on autologous arterial emboli in the cerebral circulation of rabbits, Stroke 25 Ž1994. 651–656. w7x N. Heye, A. Campos, S. Sampaolo, J. Cervos-Navarro, Morphomet´ rical evaluation of triflusal in brain infarction, Acta Neurochir. 57 Ž1993. 53–55. w8x N. Jiang, R.L. Zhang, H. Chen, M. Chopp, Anti-CD11b monoclonal antibody reduces ischemic cell damage after transient Ž2 h. but not after permanent MCA occlusion in the rat, Neurosci. Res. Commun. 15 Ž1994. 85–93. w9x Q. Jiang, M. Chopp, Z.G. Zhang, J.A. Helpern, R.J. Ordidge, J. Ewing, P. Jiang, B.A. Marchese, The effect of hypothermia on transient focal ischemia in rat brain evaluated by diffusion and perfusion-weighted NMR imaging, J. Cereb. Blood Flow Metab. 14 Ž1994. 732–741. w10x K. Overgaard, T. Sereghy, G. Boysen, H. Pedersen, Reduction of infarct volume by thrombolysis with rt-PA in an embolic rat stroke model, Scand. J. Clin. Invest. 53 Ž1993. 383–393. w11x K. Overgaard, T. Sereghy, G. Boysen, H. Pedersen, S. Høyer, N.H. Diemer, A rat model of reproducible cerebral infarction using thrombotic blood clot emboli, J. Cereb. Blood Flow Metab. 12 Ž1992. 484–490. w12x S.M. Papadopoulos, W.F. Chandler, M.S. Salamat, E.J. Topol, J.C.

w13x w14x

w15x w16x w17x

w18x

w19x

w20x

w21x

w22x

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