Somatostatin-like neurons are expressed in fetal neocortical homografts in adult rat spinal cord

Brain Research, 374 (1986) 147-152 Elsevier

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BRE 11729

Somatostatin-Like Neurons Are Expressed in Fetal Neocortical Homografts in Adult Rat Spinal Cord JERALD J. BERNSTEIN 1,2and JAMES R. CONNOR l 1Laboratory of Central Nervous System Injury and Regeneration, Veterans Administration Medical Center, Washington, DC 20422 and Departments of Physiology and 2Neurosurgery, George Washington University School of Medicine, Washington, DC 20037 (U.S.A.) (Accepted October 9th, 1985) Key words': central nervous system transplant - - paraplegia - - spinal cord - - somatostatin - - regeneration - - immunohistochemistry

Fetal central nervous system homografts to adult spinal cord are considered a potential aid for recovery of function after paraplegia. This study utilizes somatostatin (SOM) immunohistochemistry to study the organization of an embryonic day 14 (El4) neocortical homograft into the spinal cord of an adult host over 6 postoperative months. Although the El4 homograft does not contain SOM-positive cells, SOM-reactive neurons are expressed by 30 days postimplantation and are still present in 6-month-old homografts. SOM-immunoreactive neurons are bitufted or multipolar and have dendrites that are confined to the graft. The homograft contains SOM-immunoreactive axons entering and/or exiting from lamina II in the host dorsal horn and SOM-positive homografted neurons send axons into the host ventral columns. These data show that the SOM peptide neocortical phenotype is preserved in homografts to spinal cord but there is anatomical host-homograft integration. INTROD UCTION Fetal homo- and xenografts have been successfully transplanted into the brain of rodents6,17,28, 36. These grafts contain neurons which grow cell processes into the p a r e n c h y m a of surrounding brain. This is especially the case if the structure to be innervated by the graft has been d e n e r v a t e d by a previous drug or mechanical lesion. The neurons in fetal CNS grafts that show the most vigorous growth are m o n o a m i n e r gic nerve fibers which have been shown to actively regenerate in vivo after lesion6. In addition, homografts and some xenografts have been shown to result in the return, toward normal, of b e h a v i o r lost by mechanical or chemical lesiont4, 25. Homografts have been t r a n s p l a n t e d in the spinal cord of rodentsZ-5,vA0,tl,t3,18, 29-31. Neurons, glia and e p e n d y m a l cells 2 have been d e m o n s t r a t e d in these grafts at the light and electron microscopic level. The specification of neuronal types in spinal cord transplants have shown that specific classes of neurons can arise in these grafts at times similar to n o r m a l development. Vasoactive intestinal p o l y p e p t i d e (VIP)-im-

munoreactive neurons are expressed in neocortical homografts in the spinal cord of an adult rat host by 30 days postimplantation (DPI) and survive for at least 6 months 10,1~. These results show the preservation of homograft neocorticai p h e n o t y p e (since VIP was not present in e m b r y o n i c day 14 (E14) neocortex) by the expression of this p e p t i d e in grafted cortical neurons in an ectopic location. Immunohistochemistry can define populations of neurons with similar morphological and biochemical features and is a proven tool for determining if embryonic tissue develops normally when transplanted into adult host spinal cord1°, it. Somatostatin ( S O M ) immunohistochemistry will be utilized in the following experiments to evaluate: (1) if fetal neocortical homografts i m p l a n t e d into adult rat spinal cord retain the precursors of S O M - i m m u n o r e a c t i v e neurons in the transplant in their new environment; (2) when SOM will be expressed in its new environment; (3) if S O M neurons integrate with the host; and (4) if there are host-related trophic or other influences that affect the p h e n o t y p i c expression of biochemical or morphological constituents of homografts or will the

Correspondence: J. Bernstein, Veterans Administration Medical Center (151Q), 50 Irving St. N.W., Washington, DC 20422, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. A: all light micrographs from sections immunoreacted for SOM. Layers III-V in normal differentiated cortex dt~nonstrating bitufted (arrow) and multipolar (arrow head) SOM-immunoreactive neurons, B: by 30 DPI, homografts could ~ccup3 nat~sl of the host gray matter and grow into the ventral columns (arrow: cresyl violet counterstain). C, D: differentiating S()M-immunoreactive neurons (arrows) at 30 DPI in the homograft (cresyl violet c~mnterstain),

149 graft develop as cerebral cortex despite its ectopic location 10. MATERIALS AND METHODS Timed-pregnant Sprague-Dawley female rats (Zivic-Miller Labs.) were anesthetized with Chloropent (180 mg/kg) on successive days and two pups were removed by caesarean section from the same uterine horn on embryonic days 14 (El4) and 15 (El5). The embryo was placed in a cold complete Tyrode-Ringer's solution. The cerebral cortex was dissected free and transferred to a second dish of cold Tyrode's solution. A 0.5 × 1.0 mm piece of embryonic cerebral cortex was aspirated into a 26-gauge needle attached to a 50 ~1 Hamilton syringe. The aspirated embryonic cortex was pressure-injected, subdurally, on the left side between the dorsal horn and column at the level of the sixth thoracic vertebrae (T6) of host adult (300 g) Sprague-Dawley male rats (anesthetized, Chloropent 180 mg/kg) (see ref. 5 for details). After implantation, gelfoam was placed over the exposed spinal cord and the wound closed. All hosts received a 0.1 ml (i.m.) injection of Longicil (Fort Dodge Labs.) postoperatively. Two adult rats were subdurally injected with complete Ringers solution only at T6 to serve as sham-operated controls. Host rats (4 in each group) were allowed to survive 30, 45 days and 3, 4 and 6 months after the implantation. The sham-implanted rats were utilized 45 days after the operation. Pups delivered by the same mother that provided the embryos for the transplant were used as normal developmental controls (8, 20, 23 or 30 days postnatally). To collect the tissue, the rats were anesthetized with Chloropent (180 mg/kg) and perfused through the ascending aorta with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The spinal cord was exposed and the region containing the implant removed and sectioned at 50 ~m on a Lancer vibratome. The brains of the control pups were removed and sectioned coronally at 50~m. Sections were rinsed in 0.1 M PBS and pretreated in NaIO 4 (0.01 M) NaBH 4 (10 mg/ml), 5% DMSO and goat serum (1:30). The antibody was directed against SOM (ImmunoNuclear Corporation) and generated in rabbit. Sections were incubated over-

night in a PBS solution containing anti-SOM (1:1000) and 0.3% Triton X-100 control sections were incubated in rabbit serum instead of anti-SOM, but were otherwise treated identically to the test sections. The immunoperoxidase method of Sternberger37 was used to visualize the sites of the antibody reaction. Sections were treated with an anti-rabbit IgG fraction prepared in goat (1:20; Miles Lab.) and incubated in peroxidase-anti-peroxidase (1:30) prior to treatment with a 3',3-diaminobenzidine solution for 30 min. The brown precipitate at the reaction site was intensified by osmication. The sections were mounted on gelatin-coated slides, dehydrated and coverslipped. Some sections were stained with cresyl violet prior to dehydration to aid in identification of the implant. RESULTS SOM-immunoreactive neurons were located in layers I - V I of the cortex of normal controls. In general, these neurons were in two morphological configurations, bitufted or multipolar (Fig. 1A). There were no SOM-positive cells in El4 neocortex. At 30 DPI the homograft could occupy the entire gray matter and grow into the ventral columns (Fig. 1B). There are poorly differentiated SOM-immunoreactive neurons with few cell processes (Fig. 1C, D). The somata are round and are grouped in clusters of 2-5 cells. By 45 DPI, the SOM-immunoreactive neurons have differentiated and resemble the bitufted and multipolar neurons of normal cortex (Fig. 2A). The dendritic trees of the bitufted cells are polar and branch into many dendritic processes resulting in the tufted appearance of the dendrite. The multipolar neurons have 3-5 multiple-branched dendritic processes. At 3-6 months postimplantation bitufted and multipolar SOM-immunoreactive neurons are clustered in the homograft (Fig. 2B). Some SOM-positive neurons, contained in the homograft, have grown into the ventral columns and juxtapose ventral horn motoneurons in the host. At no time period were dendrites of homografted neurons observed leaving the graft. However, at all time periods, axons from SOM-immunoreactive neurons could be traced into the ventral columns (Fig. 2B). In addition, SOM-immunoreactive axons are

150

Fig. 2. A: differentiated bitufted (arrow) and muttipolar (arrow head) SOM-immunoreactive neurons m the homogral~ a ~,45 I)H, SOM-immunoreactive processes course through the homograft (open arrow). B: SOM-immunoreactive neuron (arrow) and cell process (arrow head; most probably a dendrite) in the ventral columns 6 months after homograft implantation. Fhere arc- SOM-immunoreactive axons (open arrow) from grafted SOM neurons (arrow) that enter the ventral columns of the hosl entering and/or exiting the homograft from R e x e d lamina II in the dorsal horn. DISCUSSION These data show that S O M - l i k e immunoreactivity is expressed in E14 neocortical homografts in the thoracic spinal cord of adult rats. A l t h o u g h S O M - c o n taining neurons in the homograft differentiate later than in normal cortex z5 and have variable m o r p h o l o gies in other parts of the brain 19,23, they are recognizable as cortical S O M - c o n t a i n i n g neurons 9,~5, 20,22,27~29.33 in the homograft. The h o m o g r a f t e d neocortical S O M - i m m u n o r e a c tive neurons are multipolar and bitufted as two gen-

eralizable morphological categories. These morphologies are normal for S O M neurons in layers I - V 1 of the c o r t e x 9,~5,'-2,29,33. The E 14 homograft was not positive for the S O M antibody ~,1~J,11,15,25.35 which agrees with published findings that the peptide is not expressed until a p p r o x i m a t e l y birth to about two weeks after birth in the visual cortex 27. H o w e v e r , the peptide is expressed in the neocortical homograft in the spinal cord and also in cortical to cortical homografts in rats LS. S O M and V I P are both expressed in neocortical homografts to the spinal cord demonstrating the preservation of p h e n o t y p e for the expression of at least these two peptides. The S O M response, robust and expressed in vitro from cell suspensions of spinal cord 32, is found in 3 distinct forms 21.2~ (SS-14; two,

151 perhaps precursor forms SS-28), has two types of

axons in the spinal cord16. Thus, axons from grafted

neuronal receptors in brain 39 and is axoplasmically

neurons have entered channels for fiber distribution to the entire spinal cord. The sections i m m u n o -

transported 26. In addition, SOM is coextensive with neuropeptide yg, noradrenalineZ2, and, in some neurons, y-aminobutyric acid 33. The transplanted neurons in the homograft should also contain these substances. In normal spinal cord SOM-positive axons are numerous in Rexed laminae I - I I I , become decreasingly a b u n d a n t in the ventral horn s and travel in dorsal, lateral and ventral columns8A6,21,22,24,34,35. SOM-con-

reacted for SOM were followed by VIP immunoreaction in the transplants which have been reported on in another study 1°,11. Both SOM- and VIP-containing grafted neurons juxtapose host ventral horn neurons. VIP is coextensive with acetylcholine2z. SOM is coextensive with neuropeptide yg.2o, noradrenaline22 and occasionally G A B A 33. If juxtaposed host ventral

taining n e u r o n s are located in the dorsal horn and in

horn neurons and the homografted SOre and VIP neurons synapse, this can result in many new trans-

the intermediolateral nucleus of the rat spinal cord 12. 16.21.34. Dorsal root ganglia also contain SOM neu-

mitter-modulator combinations and interactive possibilities in the host spinal cord.

rons whose axons pass into the dorsal horn. Dorsal rhizotomy results in the p e r m a n e n t loss of the peptide in the cat spinal cord 38.

ACKNOWLEDGEMENTS

SOM neurons in the homograft juxtapose host ventral horn m o t o n e u r o n s . This is a position not normal for spinal SOM neurons. In spite of this abnormal location, S O M - i m m u n o r e a c t i v e axons enter the ventral columns from these grafted SOM-positive neurons. This is a normal mode of passage of SOM

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Supported by the Veterans Administration. The authors thank Dr. Y. Tang, a visiting scholar from the Central Laboratory, Beijing College of Traditional Chinese Medicine, Beijing, The Peoples Republic of China, for aid with the transplants and Mr. D. Chandler for aid with the immunohistochemistry.

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