Neuroscience Letters, 147 (1992) 21-24 21 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 09080

Increase in synaptophysin immunoreactivity following cortical infarction

R. Paul Stroemer a'c, T h o m a s A. K e n t b and Claire E. Hulsebosch ax

Departments of aAnatomy and Neurosciences, bNeurology and the "Marine Biomedical Institute, University of Texas Medical Branch, Galveston, TX 77550-2772 (USA)

(Received 20 April 1992; Revised version received 17 August 1992; Accepted 19 August 1992)

Key words: Synaptophysin; Cerebral ischemia; Plasticity; Synaptogenesis; Immunohistochemistry; Middle cerebral artery

Plasticity in the central nervous system has been demonstrated using lesions of the hippocampus and rhinal cortex but has not been well studied after cerebral ischemia. Focal cerebral ischemia creates an area of infarction that is surrounded by neuronal tissue that may respond to nearby damage by creating new synapses. To determine if synaptogenesis occurs, antibodies to synaptophysin, a calcium-binding protein found on synaptic vesicles, were used with immunohistochemical techniques to assess the level of synaptophysin immunoreactivity as a measure of changes in the number of synapses. Cerebral ischemia was induced in hypertensive rats by permanently occluding the distal middle cerebral artery and ipsilateral common carotid artery. After 2 months recovery, the animals were perfused and the brains removed for immunohistochemical processing and evaluation. When comparing the cortex surrounding the infarcted area to similar areas on the contralateral side of the brain, the infarcted side had increased levels of anti-synaptophysin like activity that are statistically significant. We hypothesize that this increase in synaptophysin immunoreactivity is due to an increase in synapses in the cortex surrounding an area of infarction and supports the hypothesis of plasticity in the cortex following cerebral infarction.

The existence of central nervous system (CNS) plastic- ity after trauma is a controversial issue [4, 8-10]. How- ever, in at least one CNS region, the hippocampus, exten- sive studies have provided evidence that plasticity does occur after various ablation models [8, 9]. This plasticity establishes the restoration of function by changes in elec- trophysiological, anatomical and biochemical parame- ters [9]. Consequently, it is reasonable to predict that these events occur following trauma in other regions of the CNS, which provide for plasticity after neural trauma. These processes and their long-term effects have not been well researched in the cerebral cortex following cerebral ischemia.

In the period following cerebral ischemic infarction, massive neuronal death will result in regions of denerva- tion which could provide a stimulus for undamaged neu- rons to sprout and establish new synaptic connections. If an increase in the number of synapses results, this should be accompanied by an increase in the synaptic vesicle population and synaptic vesicle proteins in this region. It is the objective of this study to use quantitative immu- nohistochemical techniques as an indicator of the num-

Correspondence." R.E Stroemer, Department of Anatomy and Neuro- sciences, H-43, University of Texas Medical Branch, 200 University Blvd., Galveston, TX 77550-2772, USA.

ber of synapses in areas of the cortex surrounding is- chemic damage following middle cerebral artery occlu- sion using the methods of Chen and Brint [2, 7]. These methods produce a well circumscribed reduction in per- fusion with resulting infarction that is consistent in size and placement in spontaneous hypertensive rats [1, 2]. The availability of a reliable antibody to synaptophysin, a calcium-binding protein found on synaptic vesicles makes this study possible.

Four non-fasted male spontaneous hypertensive rats (260-300 g) were anesthetized with halothane (4% induc- tion/1 % maintenance) placed on a heating pad, and given an antibiotic (streptomycin, i.p., 0. l0 ml). The right com- mon carotid sheath was exposed by a ventral midline incision, the sheath removed, the vagus nerve separated, and the common carotid was permanently ligated with 4-0 suture. The rat was placed in a head clamp and an incision was made between the right eye and ear and the underlying temporalis was excised. A saline drip was started on the exposed skull, and a craniotomy was per- formed 1-2 mm rostral to the squamosal fusion. The un- derlying dura was penetrated and the middle cerebral artery was permanently ligated with a 10-0 suture proxi- mal to the frontal branch. The wounds were then sutured shut with 4-0 prolene and the animal was taken off anes- thesia and allowed to recover in a cage on a heating pad

22

Fig. 1. Line drawing of 60-/am coronal slice at the level of the corpus

callosum and optic chiasm. Insets are micrographs of immunoreact ion

product for anti-synaptophysin-like activity with no other counterstain.

Note the absence of immunoreact ion product in white matter and the

expansion of lateral cerebral ventricles (v) and cavitation of infarcted

area (arrowhead). Bar - 1 ram.

for 24 h. If an animal survived 12 h after surgery there was no subsequent mortality. Functional damage could not be detected by gross motor and sensory observa- tions. Tests for other behavioral dysfunctions are cur- rently being evaluated. Antibiotics (Streptomycin, i.p., 0.10 ml, 150 mg/ml/day) were given for 2 days after sur- gery. The animals were then housed two to a cage.

After 2 months, the animals were anesthetized with sodium pentobarbital, perfused transcardially with 4% paraformaldehyde fixative, brains were removed, taken through graded sucrose solutions in fixative up to 30%, blocked and embedded in OTC, frozen and stored at -70°C. Cryosections were cut at 60 / lm thickness and placed on coated microscope slides (1% gelatin, 0.1% po- tassium chromium sulfate) and allowed to air dry for 2 days. The sections were immunostained on slides using antibodies to synaptophysin (Boehringer Mannheim) at a dilution of 1:500. The immunoreaction was visualized using standard HRP/DAB techniques and intensified with nickel ammonium sulfate and cobalt chloride. Opti- cal density measurements were made using a Quantex image analyzer system. A standard square of tissue 0.2 × 0.2 mm was used to measure radians (light trans- mission) on a scale of 1-200 of 10 regions, 5 medial and 5 lateral, in the cortex adjacent to the ischemic damage. Readings were made in a line perpendicular to the sur- face of the cortex. For comparison, similar measure- ments were taken from analogous locations in the same

slice in the contralateral non-ischemic cortex. Background levels of anti-synaptophysin-like im- munoreactivity were determined by measuring areas of the corpus callosum. The corpus callosum was chosen because synaptophysin levels are low due to an absence of nerve terminals. These background levels were sub- tracted from the cortical measurements and normalized to a scale of 1-100 where 0 equals 0% of transmitted light blocked and 100 equals 100% blockage of transmitted light allowing the establishment of percent optical densi- ties. Optical densities of the ischemic side were then com- pared to the contralaterat non-ischemic side. Results were analyzed for significance using a paired Student's t-test within each animal and a Student's t-test for a group comparison with P < 0.05 as the level of confi- dence.

At the time of sacrifice the region supplied by the oc- cluded middle cerebral artery, principally the parietal and occipital cortex, was absent on the occluded side. By comparison, the contralateral side appeared undamaged (Fig. 1). Additionally, it should be noted that the histol- ogy of the hippocampus and other subcortical structures appeared normal in thionin-stained, ammoniacal silver- stained and synaptophysin-immunoreacted sections. Both of the lateral and the third ventricles appeared en- larged relative to non-infarcted animals.

Synaptophysin-like immunoreactivity was demon- strated in the cortex and hippocampus of both hemi- spheres with no immunoreactivity demonstrated in the corpus callosum. In general, the synaptophysin-like im- munoreactivity was diffuse within grey matter structures. The reaction product appeared darker in regions near the ischemic cortex and focal regions on the contralateral cortex. It should be noted that the intensity of im- munoreaction products demonstrate considerable varia- tion from animal to animal but were consistent within each animal and within each section. The optical densi- ties of the cortical regions of the ischemic side compared to similar regions on the contralateral side are displayed

TABLE 1

MEANS _+ S T A N D A R D DEVIATIONS OF THE OPTICAL DEN-

SITIES S H O W N AS P E R C E N T OF T R A N S M I T T E D LIGHT FOR

EACH OF THE 4 I N D I V I D U A L BRAINS A N D ALL 4 MEANS C O M B I N E D _+ S T A N D A R D DEVIATION

Ipsilateral side (%) Contralateral side (%)

Brain 1 * 21.0_+8.0 7.7_+3.8

Brain 2* 13.2_+2.3 4.2_+ 1.6 Brain 3* 23.7_+7.4 16.9+4.0

Brain 4* 21.1 _+7.5 12.6_+6.2

Mean** 19.8_+4.5 10.4_+5.6

*P<0.01 paired Student 's t-test. **P<0.05 Student 's t-test.

23

I I s c h e m i c [--- ' l Control

3O

c d~ C3

2 0

(a

1 2 3 4 Mean

Animal

Fig. 2. Histogram of optical densities + standard deviations of the cor- tex ipsilateral and contralateral to the ischemic damage for each of the 4 rats used in the present study and the mean of the means for all 4 rats.

*P < 0.01 paired Student's t-test. **P < 0.05 Student's t-test.

in Table I. Note that the optical density readings are different between sides for each animal and this differ- ence is statistically significant using a paired Student's t-test (all brains P < 0.01, brain 1: P < 0.0001, brain 2: P < 0.00001, brains 3 and 4: P < 0.01). The readings from the 4 animals used in the study are averaged to- gether (Table I) and demonstrate a statistically signifi- cant difference using a Student's t-test (P < 0.05). These data are displayed in histogram form in Fig. 2.

Synaptophysin, a presynaptic vesicle protein (M r 38,000), is found in virtually all nerve terminals. Levels of synaptophysin within the terminal are believed to re- main constant along with several other vesicle proteins due to the recycling of vesicle material in the nerve termi- nal [19, 21, 22]. Methods developed by Masliah et al. [16] allow the estimation of increases or decreases in synaptic numbers using synaptophysin immunostaining and are now used by others in the fields of anatomical remodel- ing and neural development [3, 5, 15-18, 23].

Although, the existence of anatomical sprouting and synaptogenesis in the central nervous system after trauma is a subject of controversy, several lines of evi- dence using behavioral and trauma paradigms support the hypothesis of anatomical plasticity in the cortex. Cortical thickening and increases in dendritic branching in adult rats following exposure to an enriched environ- ment and the converse, cortical thinning and decreases in dendritic branching as a result of an isolated environ- ment have been reported [12]. Similar work using elec- tron microscopy techniques coupled with stereology, demonstrated decreases in neuronal numbers but no change in total synaptic numbers as a result of an en- riched environment [20]. An increase in dendritic branch- ing following maze training has also been observed [6]. Increased dendritic branching visualized with Golgi

staining techniques after hemidecortications and unilat- eral motor and bilateral medial frontal cortex lesions in rats has been reported and in some cases is accompanied by behavioral recovery [13, 14]. Changes in dendritic branching could help promote long term changes in fuc- tional maps surrounding focal ischemic lesions. Such changes in functional maps have been reported using electrophysiological tracking in primates [11]. Conse- quently the concept of cortical plasticity in response to trauma and behavioral modifications is becoming well documented and provides a basis for the hypothesis of an increase in synaptic numbers following focal ischemic in- farction.

The increase in synaptophysin-like immunoreactivity supports the hypothesis that an increase in the number of synapses has occurred in the region adjacent to ischemic lesion. This is in agreement with reports of an increase in dendritic branching as seen by others following neural trauma [8, 9]. An increase in dendritic branching would allow for synaptogenesis by providing more surface area and thus greater potential for axo-dendritic connections for neural communication. Ultrastructural studies are underway to determine the classification of the new syn- apses. Lesions of injury to the CNS must signal an upreg- ulation of the necessary proteins if synaptic remodeling is to occur. The understanding of the molecular basis for injury-induced neurite growth and synaptogenesis is apt to be important regarding restoration of function of the damaged CNS, but remains to be elucidated.

This work is supported by NS 07185, NS 11255, NS 01217, RR 03979 and Bristol Myers-Squibb.

1 Bradley, R.H., Kent, T.A., Eisenberg, H.M. and Quast, M.J., Mid- dle cerebral artery occlusion in rats studied by magnetic resonance imaging, Stroke, 20 (1989) 1032-1036.

2 Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. and Pulsinelli, W., Focal brain ischemia in the rat: methods for reproducible neocorti- cal infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries, J. Cereb. Blood Flow Metab., 8 (1988) 474-485.

3 Brock, T.O. and O'Callaghan, J.P., Quantitative changes in the syn- aptic vesicle proteins synapsin 1 and p38 and the astrocyte-specific protein glial fibrillary acidic protein are associated with chemical- induced injury to the rat central nervous system, J. Neurosci., 7 (1987) 931 942.

4 Brown, P.B., A reassessment of evidence for primary afferent sprouting in the dorsal horn. In L.M. Pubols and B.J. Sessle (Eds.), Effects of Injury on Trigeminal and Spinal Somatosensory Systems, Liss, New York, 1987.

5 Cabalka, L.M., Ritchie, T.C. and Coulter, J.D., Immunolocaliza- tion and quantitation of a novel nerve terminal protein in spinal cord development, J. Comp. Neurol., 295 (1990) 83-91.

6 Chang, F.F. and Greenough, W.T., Lateralized effects of monocu- lar training on dendritic branching in adult split-brain rats, Brain Res., 232 (1982) 283-292.

24

7 Chen, S.T., Hsu, C.Y., Hogan, E.L., Maricq, H. and Balentine, J.D., A model of focal ischemic stroke in the rat: reproducible ex- tensive cortical infarction, Stroke, 17 (1986) 738-743.

8 Cotman, C.W., Specificity of synaptic growth in brain: remodeling induced by kainic acid lesions, Prog. Brain Res., 51 (1979) 203-215.

9 Cotman, C.W. and Nieto-Sampedro, M., Brain function, synapse renewal, and plasticity, Annu. Rev. Psychol., 33 (1982) 371~,01.

10 Finger, S. and Stein, D.G., Brain Damage and Recovery Research and Clinical Perspectives, Academic Press, Orlando, 1982.

11 Jenkins, W.M., Merzenich, M.M. and Recanzone, G., Neocortical representational dynamics in adult primates: implication for neu- ropsychology, Neuropsychologia, 28 (1990) 573 584.

12 Juraska, J.M., Greenough, W.T., Elliott, C., Mack, K.J. and Berkowitz, R., Plasticity in adult rat visual cortex: an examination of several cell populations after differential rearing, Behav. Neurol. Biol., 29 (1980) 157 167.

13 Kolb, B. and Gibb, R., Sparing of function after frontal lesions correlates with increased cortical dendritic branching: a possible mechanism for the Kennard effect, Behav. Brain Res., 43 (1991) 51-56.

14 Kolb, B., Sparing and Recovery of Function. In B. Kolb and R.C. Tees (Eds.), The Cerebral Cortex of the Rat, MIT Press, Cam- bridge, 1990 pp 537-561.

15 LeClerc, N., Beesley, P.W., Brown, 1., Colonnier, M., Gurd, J.W., Paladino, T. and Hawkes, R., Synaptophysin expression during synaptogenesis in the rat cerebellar cortex, J. Comp. Neurol., 280 (1989) 197-212.

16 Masliah, E., Terry, R.D., DeTeresa, R. and Hansen, L.A., lmmu- nohistochemical quantification of the synapse-related protein syn- aptophysin in AIzheimer disease, Neurosci. Lett., 13 (1989) 234 239.

17 Masliah, E., Terry, R.D.. Alford, M. and DeTeresa, R., Quantita- tive immunohistochemistry of synaptophysin in human neocortex: an alternative method to estimate density of presynaptic terminals in paraffin sections, J. Histochem. Cytochem., 38 (1990) 837-844.

18 Masliah, E., Fagan, A.M., Terry, R.D., DeTeresa, R., Mallory, M. and Gage, F.H., Reactive synaptogenesis assessed by synapto- physin immunoreactivity is associated with GAP-43 in the dentate gyrus of the adult rat, Exp. Neurol., 113 (1991 ) 131-142.

19 Sudhof, T.C. and Jahn, R., Proteins of synaptic vesicles involved in exocytosis and membrane recycling, Neuron, 6 (1991) 665 677.

20 Turner, A.M. and Greenough, W.T., Differential rearing effects on rat visual cortex synapses. I. Synaptic and neuronal density and synapses per neuron, Brain Res., 329 (1985) 195 203.

21 Walaas, S.I., Jahn, R. and Greengard, E, Quantitation of nerve terminal populations: synaptic vesicle-associated proteins as mark- ers for synaptic density in the rat neostriatum, Synapse, 2 (1988) 516 520.

22 Wiedemann, B. and Franke, W.W., Identification and localization of synaptophysin and integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles, Cell, 41 (1985) 1017 1028.

23 Wright, D.J., Ritchie, T.C. and Coulter, J.D., Distribution and de- velopmental expression of the nerve terminal protein NT75 in the rat cerebellum, J. Comp. Neurol., 304 (1991) 530-543.