0306-4522/90 $3.00+0.00 Pergamon Press plc c‘ 1990 IBRO

iVeurosc;mce Vol. 35.No. 3,pp.559-575, 1990 Printed in Great Britain

MAINTENANCE OF LOCAL CEREBRAL BLOOD FLOW AFTER ACUTE NEURONAL DEATH: POSSIBLE ROLE OF NON-NEURONAL CELLS C. IADECOLA, S. P. ARNERIC, H. D. BAKER, J. CALLAWAY and D. J. REIS Cornell

Division of Neurobiology, Department of Neurology and Neuroscience, University Medical College, 411 East 69th Street, New York, NY 10021, U.S.A.

Abstract-In brain, a major factor regulating local perfusion is local neuronal activity. However, we have recently discovered that, in rat, five days after selective neuronal destruction in the parietal cortex by local microinjections of the excitotoxin ibotenic acid, local cerebral blood flow, within the lesion, remains in the normal range. We studied whether proliferating non-neuronal ceils and/or local changes in microvascular density participate to maintain local cerebral blood flow. Rats were anesthetized (halothane l-3%), ibotenic acid (IO pg in 1 ~1) was locally microinjected in a restricted region of the parietal cortex, and animals were allowed to recover. Three, five, seven, I I, 30 days later local cerebral blood flow was measured autoradiographically under chloralose anesthesia (40 mg/kg, s.c.) by the [‘4C]iodoantipyrine technique. Cellular density or microvascular area were determined on sections stained with Thionine or processed for the endothelial marker alkaline phosphatase, respectively. Local neurons were destroyed by 24 h after microinjections of ibotenic acid. However, from three to 11 days after lesion local cerebral blood flow was unchanged (P > 0.05; n = 5), thereafter declining so that by 30 days blood flow was 48 k 6% of control (P < 0.05; n = 5). Cellular density increased within the lesion by 17.5-fold at seven to I I days (P < 0.01) and declined to a 1 I.7-fold elevation above control at day 30 (P < 0.01). New cells consisted of macrophages, endothelium and glial fibrillary acidic protein-positive astrocytes. The microvascular area increased 4.2-fold from three to 1I days (P < 0.01). The patency of the presumably newly formed vessels was determined by the presence of intravascular red blood cells, which were revealed histochemically. The area occupied by red blood cells within cerebral microvessels, in contrast to microvascular area, did not increase until seven days after lesion, reaching a 3.2-fold increase at I I days. Thus within the lesion, local cerebral blood flow remains constant during the phase in which cellular and microvascular density increases. The presumably newly formed vessels cannot contribute to maintain local cerebral blood flow since during this phase they are not patent; rather patency develops coincident with the decline in local cerebra1 blood flow. We conclude that non-neuronal cells, most likely activated macrophages, may be an important factor regulating local cerebral perfusion, after acute neuronal death.

anesthesia3’ or brainstem hypothermia, ” barbiturate stimulation,36 then ICBF will change in the same direction and proportionally. Although recent observations in rat3’ have indicated that there are conditions in which ICBF can vary independently of ICGU, the concept of the close correspondence or “coupling” between flow and metabolism remains generally valid.3’ It is also widely believed that a major determinant of ICGU is the activity of local neurons while nonneuronal cells, having lower metabolic demands, do not contribute substantially to the local metabolism in a region. ” However, we have recently observed in rat that, five days after elimination of local neurons in a small area of the cerebral cortex by local microinjection of the excitotoxin ibotenic acid, ICBF does not change.22 This finding is very surprising and suggests the possibility that cells other than neurons may, under certain conditions, contribute to the maintenance of local cerebral perfusion. In the present study we have examined the contribution of neuronal and non-neuronal cells to the local perfusion of a restricted region of the cerebral

It is well established that, in brain, one of the major factors regulating local cerebral blood flow (ICBF) is local metabolic activity, which is closely related to local cerebral glucose utilization (lCGU).49 This concept, introduced by Roy and Sherrington46 and confirmed in the past two decades with the introduction of autoradiographic techniques for measuring ICBF and lCGU,43.47.49is based, mainly, on two lines of evidence. Firstly, in “resting” brain there is a correspondence between values of flow and metabolism, so that regions with high ICBF have high ICGU while regions with low ICBF have low lCGU.43,44 Secondly, if metabolism is increased, as for example, during mental activity,44 in stress,” hyperthermia,’ excitation of neural pathways’O and seizures,3s or if it is decreased as, for example, during Ahhrrc~iations: GFAP, glial fibrillary acidic protein; ICBF, local cerebra1 blood flow; ICGU, local cerebral glucose utilization; MVA, microvascular area; paCO,, arterial partial pressure of COz; paOz, arterial partiai pressure of 02; PAP. peroxidaseeantipcroxidase; PI, patency index; RBC, red blood cells; RBCA, red blood cells area; SMI, primary sensory cortex. 559

cortex nated

in which

local

neurons

were

selectively

by using ibotenic acid. Preliminary

portions

of this study

have

EXPERIMENTAL

been

elimi-

reports of

presented.”

PROCEDLiRES

Studies were performed on 4X male Sprague-Dawley rats (Hilltop Lab. Animals Inc.. Scottsdale. PA. U.S.A.) weighing 300 3XOg. maintained in a thermally controlled (20 C). light-cycled (07:OO on. 19:OO olt‘) environment and fed laboratory chow trrl /ihi/tu?r.

Neurons in a restricted cortical area were destroyed by local cortical microinjection of the excitotoxin ibotenic acid.‘2.‘x Rats were anesthetized with halothanc (I 3% in 100% oxygen) and placed on the stereotaxic frame (Kopf). The calvarium over the frontoparietal region was exposed and burr holes. 3 mm in diameter, were placed with a dental drill bilaterally in the parietal bones. 3 mm lateral and 4 mm caudal to the bregma. This area overlies the primary sensory cortex in the rat. During the procedure care was taken not to overheat the bone and not to damage the clzrrrr tmr/er. Calibrated micropipettes (tip diameter 50 75pm) were loaded with agents by vacuum and fitted to the electrode carrier of :I stereotaxic apparatus. Ibotenic acid. lO,r~g in I ~tl of 100 mM phosphate-buffered saline (pH 7.4) was loaded into the micropipettc and the micropipette inserted. at a -~ I9 angle. into a zone of the parietal region located 4.0 mm caudal and 3 mm lateral to bregma. The dura was pierced and the pipette lowered I.5 mm below the dural surface. Ibotenic acid or vehicle was then slowly delivered over a period of 5 min using the positive-pressure system of Amaral and Price.’ At the end of the injection the pipette was left in place for I min and then withdrawn. Vehicle was injected into the homotopic area of the contralateral cerebral cortex. In sham-operated controls, vehicle was injected bilaterally into the site. Wounds were infiltrated with 2”;, procaine. sutured closed. the animals removed from the stereotaxic frame and returned to their cages. One to two hours later. animals had fully recovered from the anesthesia. They were active and did not show signs 01 discomfort. One to 30 days later. rats were reanesthetized for measurement of ICBF or for morphological studies (see below).

Methods for surgical preparation of animals and for measurement of ICBF using the iodoantipyrinc technique wtith quantitative autoradiography4’ arc identical to those described in earlier publications.“‘~“’ I’ and are summarized below .Surgic~dpwpwtrtio~~.Animals were anesthetized with chloralose (40 mg;kg, s.c.) after induction with halothanc (I 29,) in 100% oxygen) administered through a facial mask. Catheters were inserted in the femoral arteries. veins

and m the trachea and the animals were placed on the stcrcotaxic frame with ear bars and mouth-piece loosely titted. One of the arterial catheters was connected to a pressurc transducer (Statham P23Db) fed into a polygraph (Grass Mod. 7D) for continuous monitoring of arterial pt-essure and heart rate. Body temperature was kept at 37 t 0.5 C by a thermostatically controlled heating lamp co%cted to a rectal probe. Animals were then paralysed with tl-tubocurarine (0.5 mg;kg, i.m.) and artificially ventilated by a mechanical ventilator (Harvard Apparatus. Mod 940). Blood gases were monitored on samples (0.2 ml) of arterial blood by a blood gas analyzer (Instrumentation I.ahoratory. Micro I3 system). and kept in physiological range. Arterial partial pressure of CO, (paC0,) was ixiiw mined in the normocapnic range (33 38 mmHg) by adjusting the stroke volume of the ventilator. Arterial partial pressure of 0: (pa02) was maintained above 100 mmHg by v’entilating the animal with 100% oxygen in order to countcract the hypoxia resulting from the atelectasis commonly seen in rats paralysed and artificially ventilated.” As ;I conscqucnce pa0, in our groups is high (Table I). Howcvcr. this is not a reason of concern in our studies since this dcgrec of hyperoxia does not affect ICBF.” ICBF was measured after blood gases were normal range and in a steady-state (Table I). Experiments usually lasted less than 3 h. .~~~,~I.SII~C~IICIII o/’ loc,ul i,crchr~I blood ,flon.. ICBF was measured using iodoantipyrine as a diffusible and inert indicator.” The brain concentration of the tracer was measured by quantitative autoradiography.?“.“’ As described in detail elsewhere.“’ 4-(iii-methyl-“C)-iodoantipyrinc in ethanol solution (New England Nuclear Corp.; specitic activity: 40 60 mCi;‘mmol) was dissolved in I ml of saline after evaporation of ethanol. Prior to infusion. animals reccivjed SO0 U hepdrin i.v. (A. H. Robins). Iodoantipyrine was infused (IO~Ci~lOOg body weight) at a constant rate for approximately 30 s through one of the femoral VCIIOU~ catheters. by an infusion pump (Harvard Apparatus. Mod. 940). Simultaneously. timed samples of arterial blood wcrc collected from the femoral arterial catheter to obtain the arterial concentration time-curve of iodoantipyrine. Forty microlitcr aliquots of blood were transferred into scinttllation vials, and. after solubilization and decoloration. vials wet-c filled with a scintillation solution (Ready Solv HP’b. Beckman) and radioactivity (dpm) determined in a scintiiIation spectrophotometer (Beckman. Mod. LS5801). At the end of the infusion period. animals were killed hy an 1.1. bolus injection of I ml of saturated KCI. The brain was rapidly removed from the skull, frozen in Freon-17 at 30 C. and mounted on a cryostat chuck with embedding medium (Lipshaw). Sections. 20-/lm-thick were cut at 200-/lrn intervals in a freezing microtome (H’I Bright), removed from the blade with a coverslip and dryed on it hot plate at 60 C. Additional sections were collected through the atma of the lesion and kept frozen. for subsequent staining for alkaline or acid phosphatase (set below). F’or autoradiography, coverslips were mounted on glass slides and the slides fitted in X-ray cassettes together with calibrated [“‘Clmethyl-methacrylate standards. Sections and standards

Table I, Mean arterial pressure, blood gases, and hcmatocrtt in anesthetized paralysed without lesions of the primary sensory cortex by ibotenic acid

rats with and

Days after lesion

I31 35.9 264 7.41

*3 j I F 39* + 0.03 50 f I 5

*P < 0.05 analysis of variance MAP. mean arterial pressure:

>

3

Sham-lesioned MAP (mmHg) paC0, (mmHg) pa0, (mmHg) PH H t ( ‘%,) II

I19 35.3 335 7.43 51

* + f * * 4

and Newman Ht. hematocrit.

6 0.9 36 0.01 I

I’1 36. I 419 7.39 51

Keuls test.

25 & 0.4 & IO f 0.01* f 0.7 6

I5 13.5&5 36.5 * 0.6 3x4 & ix 7.45 * 0.01 49 * 0.7 5

30 I%*3 36.5 f 319 * 7 46 + 50 + 5

0.5 10 0.01 0.8

Non-neuronal were expnsed on X-ray film (Kodak, SB5) and, five days later, the film developed. Alternate sections were stained by the Nissl method and were used as anatomical reference for the analysis of the autoradiograms (see below). As described in detail elsewhere,“0,‘7 autoradiograms were analysed by using a Vidicon-based image analysis system (Eyecom II) connected to a PDP I l/45 minicomputer (Digital). Autoradio~rams were digitized and the relationship between optical density, tissue concentration of iodoantipyrine (nCi/g) and ICBF (ml/100 g x min) established based upon standards on the film and the arterial timecourse of iodoant~pyr~ne.~~ Regions of interest (Table 2) were encircled by a joystick-controlled cursor and ICBF measured. For each region, readings were taken bilaterally, usually, over four consecutive sections, and averaged, independently, for right and left side, At the level of the lesion readings were taken in those sections in which the area of the lesion, identified on the adjacent Nissl-stained sections. was greatest.

cells and CBF

Meusurement qfmicrovuscular area. MVA was measured by using the image analyser with the Vidicon scanner attached to a microscope with a IO x objective. The measuring field was 0.42 mm’. The center of the area of the lesion was digitized and the grey scale level of the image was varied by the operator until background noise was eliminated. MVA was automatically computed as the percentage of the area occupied by the alkaline phosphatase-positive microvessels relative to background. For each case MVA was compared between the lesion and the contralateral (unlesioned) cortex in four adjacent sections. Values of MVA in the lesioned and unlesioned cortex were averaged independently. It is likely that the MVA in this study, as in those of others.‘9,s0 represents an overestimation of the “true” MVA as a result of tissue thickness. However, the error is uniform for all cases and minimized by the fact that the contralateral side served as controi thereby not affecting the interpretation of the results. Dcterminution

Non-neuronal cells in the area of the lesion were counted on sections (thickness 20pm), adjacent to those used for ICBF autoradiography, stained by the Nissl method. Cells (i.e, nuclei) were counted using a microscope (40 x f equipped with an ocular grid (grid dimensions: 140 x 140pm). For each animal cells were counted over three adjacent sections. For each section five fields were counted in the area of the lesion and five in the homotopic cortical area ~ont~~aterally, The average cellular density (cells/grid), for the lesion and the contralateral area, was computed by averaging the values af the fields and then the values of the sections. The area of the lesion was measured in the same fresh-frozen, Nissl-stained sections used for cell-counting. Measurements were performed by using the image analysis system with the Vidicon scanner connected to a microscope (Leitz). Firstly, the area of each picture element (pixel) was established by calibrating the system with a micrometric scale. Brain sections were digitized and the area of the lesion encircled by using a joystick-controlled cursor. The area of the encircled region was then automatically computed by the number of pixels included in the region. Measurement

of micro~~a.scukur area

Microvessels were visualized by staining sections histochemically for the enzyme alkaline phosphatase. AIkaIine phosphatase activity, virtually absent in the brain parenchyma, is highly concentrated in the endothelium of cerebral arterioles and capillaries.’ Thus. alkaline phosphatasc activity is a reliable cytological marker of brain microvessels and as such has been extensively used to study the microvascular architecture of brain in a wide variety of conditions.5,4s.s” Microvascular area (MVA) was determined on alkaline phosphatase-stained sections adjacent to those used for measurement of ICBF and for counting of cells. Alknii~ phosphatase histochemistry. Brain sections (20 pm) were stained for alkaline phosphatase by using the azo dye method with simultaneous coupling.“0 Slides were placed in buffered s~cros~~formaiin for f min, washed in H@ and incubated for 3645 min in a soiution containing 0.38% (w/v) of Fast Blue RR (coupler) (Sigma) and 0.05% of alpha-nepthyl phosphate (substrate) (Sigma) in H,O at 37 ‘C (pH = 9,0&9.4). After rinsing in H,Q, slides were placed again in buffered sucrose-forma& for 1min. rinsed in H,Q and allowed to dry. Slides were not coverslipped. The alkaline phosphatase-positive microvessels were identified as darkly-stained elongated segments. branching and forming a dense network (Fig. 2). In agreement with observations by others,5”5-M the endotheiium of large cerebral arteries, veins, venules and brain parenchyma were unstained.

561

qf‘

pyfustvi microwssels

As an index of the number of microvessels open to blood flow we measured, in a separate group of animals, the relative content of red blood cells (RBC) in brain sections stained for endogenous peroxidases as markers.’ Endugenuus pero.~~dase.~ h~~&oc~ernistr,~. Rats were deeply anesthetized with hafothane (5%) and decapitated. The brain was rapidly removed from the skull and immersed in a solution containing 0.4% paraformaldehyde and 1.25% glutaraldehyde in 0. I M phosphate buffer fpH = 7.4). After 24 h the brain was cut in 1-2 mm slices and replaced in the soiution for one week. Sections (loon were cut on a Vibratome, reacted for endogenous peroxidases using diamino benzidine as substrate,’ rinsed in 0. I M phosphate buffer and mounted on glass slides. After reaction, individual RBC loaded with reaction product were easily identified in all the segments of the cerebral vasculature. including capillaries. No activity was present in the brain parenchyma. Measurement of red biood ceils area. RBC area (RBCA) was measured in a manner identical to the measurement of MVA, by using the image analyser with the Vidicon scanner attached to a microscope. Due to the thickness of the section, there was more than one focal plane. Thus, in all cases the first focal plane was analysed. RBCA was expressed as a percentage of the area occupied by RBC relative to background. In each animal, RBCA was measured in four adjacent sections in the area of the lesion and in the contralateral cortical area and values were averaged inde~ndently. Phugocytic

acticity

in the lesion

Phagocytes were detected by staining for the lysosomal enzyme acid phosphatase.’ Sections (thickness 20 Fm). adjacent to those used for alkaline phosphatase histochemistry and KBF autaradiography, were pracessed according to a protocol identical to that used for alkaline phosphatase staining, with the only difference that the pH of the incubation solution was 5.1: .After the reaction the slides were not coverslipped. Giial .fibrillary

acidic protein immunoc_vtochemi.~tr~

Glial fibrillary acidic protein (GFAP) ~mmuno~yt~hemistry was performed on paraffin--embedded sections by using anti-GFAP antibody6 and the peroxidase-antiperoxidase (PAP) technique.6,‘Z Rats were deeply anesthetized with pentobarbital (100 mg/kg) and perfused through the heart with 150 ml of normal saline followed by 500 ml of 10% buffered formalin. The brain was removed from the skull, immersed in 10% buffered formalin for one week, cut in blocks and the bIocks embedded in paraffin using conventional procedures. Sections (tOpm) were cut from the blocks on a rotary microtome and mounted on glass slides. Selected sections were stained with hematoxylin eosin using conventional procedures, for morphological analysis. For

Table 2. Time-course

of local cerebral blood Row (mean + S.E.M.) after lesion of the primary sensory cortex by the excitotoxin ibotenic acid in rat Shamlesioned

Cerebral cortex Contralateral Lesion Front;tl

R I, R

Purietnl Auditory Visual

Basal ganglia Caudate--putamen Globus

pallidus

.~mygd~l~ Preoptic

n. magnocellularis

Thalamus Anterior Reticular Ventral Ventromedial ~ntr~l~min~r Hypothalamus

R L R L R L R I.

Ion * 100 * 69 & 61&h 88 _t x4 f lO6+1 IO6 *

R I_ R L R L R 1. R L R L

Hippocampus

Midbrain Substantia

nigra

Superior

colliculus

inferior

L R L R L K L

Il5i.s 11926 120 * 114*s lO5&7 IO4 & 97 & IO2 * 99 * 102 +

colliculus

Pontine grey Cerebellum Vermis Hemisphere Dentate

nucleus

Pans rn~dull~ Parabrachial Vestihular

R L R I.

complex complex

Nucleus reticularis

gigantocellularis

Nucleus reticularis

p~irvocellul~ris

Corpus callosum II

R L R L R L R L R 1.

Y

s 61 7 7 7

7 x 4

ICBF

(mI:lOOg

3

154 5 lo*+ IX+23 143+6* 10712 I30 + 7*t 114*7 l59&27* 12x * 13 l23+5* 10829

x min) days after lesion

5

I5

30

178f7 ll8+29 121 + 5 11314 I I3 + S 113t_3 114+4 110+4 99 2 s 45 + S

133* II 9x k 7t i22*7t 11317 107 + 4 lO6+-6 I IX & 13 IOX & 13 102i4 lOOk 6

lO3i_7 62 * 7*t 109 + 3 Ii0 _+ 5 87 + h* 8X t 6* 92,4 83 t_ 7 93 & 3 94 & 2

94 + 4 x9 * 8 68 14 66 * s 84&6i 88 * 6 98 * 5 100 i 4

115* I3 106&X 92 * 5** 90 & 7** Ilo* 10 Ill ix 126*6 I32 * 13*

9x * 9116 69 & 63+4 81 ,x 79 ) 9116 90 &

I

IiS+ 114&i? 75 * 5 68 F 4 101 F-8 99+ IO 110+9 108 cf-7

112* IO 110&X 71 is 77 * 7 VI+ S 94 + 6 107 * 7 10517 97&6 91&4 91 +4 93 & 5 94 f 5

I41 * II 133*x 96 i_ 6* 99 k 7 ll6i I3 115* 13 I21 +8t 130*9 lZl_+8 10414 III &9 124 & 7** 122*5*

IO1 f. 5 103 &4 78 * 3 78 & 3 99+4 103&S 97 & 7 99 f 5 100+4 76 2 6 77 i 5 87 & 5 xx i 7

I32 & I3 126+9 92 2 8 91 ix llS_t4 104ih l30,9 12958 112+7 lOS_th lOOi6 IlSk5* 11048

124*X 122 _th x3 _+ 6 86 k 4 94 + 6 78 2 7i 107i76 10721 10216 89I 7 92 * 7 108 * 3* l07F2

90 * 4 92 * 6 125&i 129&H 1521 16 I51 rt: IS 12119

103+x I I6 i_ IO l59f8** l54& II* 167k7 165&7 144 + 5

85 * F x5 & 6

x3 + 6 Y3 ) 2

127+x 122 &h 1542 II IS0 * IO 100 + 9

100 & 6 10657 13524t l2bF5 147 * IO lS4&8 I20 * IO

126&Z l23iZ 154& 13 152*7 l24i_”

l&i*7 too & Ii 104&1S 18829 l74& 1:

II327 109 * I2 113i8 190t 14 l80& I4

114tb 109 2 105 & 188 _t 179+

ll9+8 99 * 9 93 *6 170 + 12 l7O_t 13

I01 ) 3 91&3 8x*4 144 _t IO I.778

IO3 f S 100 + 5 197 * 9 19oi.4 103 * 7 loo+6 99 * 4 95 ri_4 47 & 4 so + 4 i

I31 _+x ll7i.s 201 + IS I97 * I4 I I4 + I2 I I5 * IO IIS& 12 IIS& IO 57 + 6 44 f 6 4

I03 i. 5+ 97 i 7 194+ 13 196& I6 94 t_ 7 94 &, I 97 f 7 96 F 6 52 & 5 51* 2 6

lxx&Y l12+6 19658 197F8 ll4& II 113+ IO 109 i_ 8 108 *9 a*4 62 f 7

X8 & 5

ht 6 4

R. right; L. left. *P