Regeneration of the Magnocellular System of the Rhesus Monkey Following Hypothalamic Lesions J. Lobo Antunes, M D , Peter W. Carmel, M D , Earl A. Zimmerman, MD, and M. Ferin, M D
The hypothalamic magnocellular system of the rhesus monkey was studied with specific immunocytochemical techniques in animals that had undergone hypothalamic lesions. The results indicate that this system maintains a regenerative capacity even when its tracts are interrupted within the hypothalamus. New neurohemal units are reconstituted from newly formed vessels within the scar as well as from preexistent blood vessels, such as perforating and pial arterioles, and the vessels of the pars tuberalis of the pituitary gland, which normally do not contain neurosecretory terminals. Antunes JL, Carmel PW, Zimmerman EA, et al: Regeneration of the magnocellular system of the rhesus monkey following hypothalamic lesions. Ann Neurol 51462-469, 1979
It is generally accepted that the central nervous system of the higher vertebrates has a limited ability to regenerate following injury [8, 161. T h e hypothalamic magnocellular neurosecretory system, which includes the supraoptic and paraventricular nuclei and their axons, constitutes an exception to this rule, and in several species section of the pituitary stalk is followed by formation in the proximal stump of a “new” neural lobe [5, 9, 12, 20, 27, 28, 3 11. This phenomenon usually becomes apparent within two to four weeks after operation and depends on the outgrowth of axons of the surviving magnocellular elements, which establish new neurovascular connections. These structures are particularly striking in preparations stained by the Gomori technique. W e have previously investigated the morphological features of the hypothalamic magnocellular neurosecretory system in the rhesus monkey using immunocytochemical techniques and specific antisera to vasopressin, oxytocin, and the associated neurophysin proteins [ 3 ] . W e have observed that the cells containing vasopressin also have so-called nicotine-stimulated neurophysin (NSN), and the cells with oxytocin also contain so-called estrogenstimulated neurophysin IESN). T h e axons of cells of the paraventricular nuclei run mostly laterally and ventrally, and when they reach the optic tract they join the fibers from the supraoptic nuclei and form a compact bundle which passes medially, ventrally, and
posteriorly behind the optic chiasm and courses under the bottom of the third ventricle into the median eminence. Some of the fibers that originate in the paraventricular nucleus terminate around the blood vessels of the zona externa of the median eminence [2]. T h e remaining fibers are arranged in longitudinal bundles that run in the infuridibular stem and terminate on the capillaries of the neural lobe. As demonstrated by immunocytochemical techniques, in intact animals the axons of the magnocellular nuclei d o not make connections with blood vessels in the pia or pars tuberdis, or with perforating hypothalamic arterioles. During investigations on the effects of various hypothalamic lesions o n endocrine function in the rhesus monkey, we found morphological evidence that regeneration of the magnocellular system may occur within the hypothalamus itself. When axons from both the paraventricular and the supraoptic nucleus are divided, the proximal segments form neurovascular connections with newly developed blood vessels within the surgical scar as well as with preexistent blood vessels which normally d o not contain neurosecretory terminals. This paper reports these observations.
From the Departmcnrs of Neurological Surgery and Neurology and the lnbritutc for the Study of Human Reproduction. Columbia University College of Physicians and Surgeons, h e w York. N Y .
Address reprint requests ro Dr Antunes, Department o f Nrurosurgery. Columbia University College of Physicians and Surgeons, 710 W 168th Sr, NKWYork, N Y 10032.
Materials and Methods The brains of 10 adult female rhesus monkeys weighing 3.5 to 4.5 kg were studied. All animals received hypothalamic lesions inflicted with a modified Halasz knife, as describecl
Accepred for publication Oct 17. 1978.
462
0364-5 134/79/050462-08$01.25 @ 1978 by J. Lob0 Antunes
previously [ 141. The purpose was to isolate the medial basal hypothalamus. which includes the ventromedial and arcuate nuclei and the most ventral extension of the supraoptic nuclei. The knife has a bayonet shape with a double-edged steel blade 1 mm wide and 6 mm high with a 3 mm radius of rotation. With the animal under pentobarbital anesthesia and with its head secured in a stereotactic frame, a burr hole was placed at the level of the coronal suture, the superior longitudinal sinus was ligated and divided, and the dura was opened. The knife was introduced vertically in the midline and the tip of the blade placed 15 mm in front and 1 or 2 mm below the external auditory meatus. The knife was then rotated 360 degrees and removed. The animals survived 2 to 28 months, after which they were anesthetized with pentobarbital and the brains fixed by cardiac perfusion with normal saline solution followed by filtered Bouin solution or 109;;;. buffered formalin. T h e calvarium was removed, the dura opened widely, and the brain immersed in the same fixative for a week. T h e hypothalamus and pituitary gland were then removed and fixed for another three to five days. T h e blocks were subsequently washed in running tap water overnight, dehydrated in graded ethanols, replaced with xylene, and embedded in paraffin. The blocks were cut in serial 6 p m coronal sections. Every tenth section was stained with cresyl violet and with hematoxylin and eosin. The immunocytochemical procedures have been described previously [2, 31. Indirect immunoperoxidase techniques using peroxidase-antiperoxidase complexes (PAP) were performed on deparaffinizec{ rehydrated sections, The following primary antisera were used: (1) rabbit antiserum to human ESN and human NSN, used at 1:5,000 and 1:500 dilutions, respectively, for 1 hour at 4°C; antisera to human neurophysins were used because neurophysins of man and rhesus monkey appear to be immunologically identical (see [31); ( 2 ) rabbit antiserum to arginine Vasopressin (7F). used at 1:1,000 for 48 hours at 4°C; and ( 3 ) rabbit antiserum to synthetic oxytocin (69.7). used at 1:250 to 1: 1,000 for 48 hours at 4°C. The secondary antiserum was sheep antirabbit globulin serum, used at 1:100 dilution for 30 minutes at room temperature. PAP was diluted at 1:1,000 for the neurophysin studies and 1:200 for use with oxytocin and vasopressin. Reaction products were formed with 3,3’-diaminobenzidine; some sections were then counterstained with cresyl violet. Controls were obtained by replacing primary antisera with nonimmunc rabbit serum or buffer. Antisera to ESN and N S N were absorbed by addition of 10 pg of purified ESN o r N S N t o 1 ml of anti-ESN (1:5,000) or anti-NSN (1:500) for 24 hours prior to use. Antisera to oxytocin and vasopressin were absorbed by adding 100 ng of synthetic oxytocin and arginine vasopressin, respectively, per 0.1 ml of 1:1,000 dilution.
Results T h e animals survived the surgical procedure without neurological deficit except for 1 monkey that became paraplegic due to injury of the anterior cerebral arteries. T h e results of the endocrine evaluation in
F I R 1 . Discrete gliotic scar containirrg a nrimber of newk formed blood ressels marking the trajeitory o f t h e kniye. Note a nrrnrber of sripranptic cells lateral l o the scar (arrow). IH&E: ~180.)
these animals have been published elsewhere [ 141. None of the animals had permanent or transient diabetes insipidus. The cuts successfully produced hypothalamic disconnections, but bilateral lesions were created in only 5 animals; the other 5 had unilateral disconnections. T h e incomplete sections were due to displacement of the wall of the third ventricle without penetration or injury by the knife on the intact side. The lesions consisted of discrete linear scars that extended from the wall of the third ventricle laterally and inferiorly to the dorsum of the optic tract. In 3 animals they reached the pial surface. T h e scar was made of glial cells and newly formed blood vessels (Fig 1). In 8 animals the nuclear structures medial and lateral to the scar were well preserved except for the paraventricular nucleus, which was invariably damaged by the shaft of the knife. In 2 other animals the knife caused extensive areas of necrosis and cavitation, which included the anterior hypothalamus, the dorsomedial and ventromedial nuclei, and part of the posterior hypothalamus and premammillary region. These lesions were unilateral in 1 monkey and bilateral in the other. They were made of multiple cysts separated by fine, fibrous septa containing newly
Antunes et al: Regeneration of Hypothalamic Magnocellular System
463
formecl branching vessels. These vessels also surrounded the necrotic areas. A number of wellpreserved neurons, many of which belonged t o the paraventricular nucleus, were seen very close to the damaged regions. 1rtrtt~utzoc:ytoibenIiiuiStudies T h e results of the imniunocytocheniical studies were similar for all antisera, indicating that cells containing vasopressinlNSN and the elements containing oxytocinlESN had similar regenerative potential. I n general, the anti-ESN and anti-NSN antisera gave more intense staining. T h e surgical scars interrupted the tracts from the paraventricular and supraoptic nuclei to a variable extent (Fig 2). In these animals, heavy accumulations of immunoreactive granular material were seen around the blood vessels of the scar, and there were also extravascular deposits with large droplets resembling Herring bodies (Fig 3 ) . T h e s e accumulations clearly demarcated the trajectory of the knife. T h e animals with necrotic lesions had extensive deposits of reactive granules surrounding new blood vessels that proliferated around areas of cavitation (Fig 4 A ) . Similar accumulations were also seen around the branching vessels within the septa forming the multiloculated areas (Fig 4 B ) . T h e s e lesions were located within the field of the paravencricularsupraopticoneurohypophyseal tract. Neurosecretory material was also seen around blood vessels in areas not crossed by magnocellular axons, such as in t h e
B
A
464
Annals ot Neurology
Vol 5
No 5
May 1970
subependymal region beneath the floor of the frontal horn of the lateral ventricle (Fig 5 ) . In the 3 animals in which the cut extended ventrally to the pial surface, there were extensive accumulations of reactive material around thc pial vessels and the pars tuberalis of the pituitary gland. I n addition, in several animals positive fibers were seen around the perforating hypothalamic arterics (Fig 6). T h e vascular deposits had a distinctive appearance, with secretory peptides either accumulated in concentric laycrs or impregnating the entire vessel wall. Frequently, beaded axons with abnormally large varicosities could be traced to these vessels, and occasionally a complete “new” neurohemal unit could be identified (Fig 6C). In many instances the interrupted axons and the areas of cystic necrosis were located very close t o magnocellular perikarya, and it did not appear that the magnitude of the regenerative response always depended o n the length of the remaining proximal segment. However, in 1 animal in which the paraventricular nucleus was completely destroyed, thcre was n o accumulation of secretory peptides in the adjacent scar. D u e to the stereotactic coordinates adopted, paraventricular axons were interrupted
B Fig 3. (A)Rear.tii,ef;bers around blood i,essels i n the scar (arrows). ( B JHigher tttu~qtiij5c~tintt oJ‘A.shnwittg headedJ5her.r drotittd blood r,rsrel.i. A lurge Jroplet oJ- ruattir.e n i ~ t e r i a l(Herrittg hodyi (arrow) is uI.ro .serti. , B o t h N S N : A x180. B X720.)
A n r u n e s er ;iI: Regc5tier;ition of Hpporhalarnic Magnoc.ellular Sysrem
465
much m o r e frequently than t h o s e froin t h e supraoptic nucleus. H o w e v e r , in 3 animals in which SUprdOptic ;ixons w e r e divided very close t o t h e perikarya o f origin, reactive deposits a r o u n d t h e newly proliferating blood vessels w e r e also f o u n d . Cell loss within t h e magnocellular nuclei did n o t exceed 20 t o 30"i of t h e total cell population. T h e perikarya of t h e retained magnocellular elem e n t s with apparently transected axons a p p e a r e d t o he slightly smaller, and their processes less n u m e r ous, than normal (Fig 7).T h e nucleus was reduced in size and t h e nucleolus less eviclent. In addition, t h e positive granular material was m o r e densely aggregated t h e n normal. Addition of' the c o r r e s p o n d i n g h o r m o n e antigens to each o f t h e respective h o r m o n e antisera abolished t h e inimunostaining. Replacing t h e antisera with buffer o r normal rabbit s e r u m revealed only t h e peroxidase activity in blood cells, and occasionally in t h e ependymal cells.
Discussion T h e hypothalamic magnocellular system has a particular ability to regenerate following injury. Desclin and Flament-Duran 113 1 r e p o r t e d that posterior pituitary transplanted into t h e hypoth,, iI a m u s c a n be reinnervated by t h e paraventricular-supraopticoneurohypophyseal tract in t h e rat. D e G r o o t [ 1 I ] m a d e electrolytic lesions in t h e cliencephaIon o f t h e rat and f o u n d in t h e damaged areas, often a t considerable distance f r o m t h e paraventricular nucleus, striictures that closely resembled t h e neural lobe; b u t t h e a u t h o r considered this finding to b e a staining artifact. O u r e x p e r i m e n t s s h o w that t h e magnocellular system retains t h e capacity to f o r m
A
B
466 Annals o f Neurology Vol 5 N o 5
May
1979
A
B
F I R 7 . Paraientricufar tixrleris in an animal i n iobirh a iinifateraffeJioti was placed. ( A )Normal side and tBi fesionerl.ride iti /be J-anie .section. (Both ESN: x 18O.i
neurovascular connections, and thus new neurohemal units, even when the axons are interrupted within the hypothalamus. Similar phenomena were recently described in the rat by Danilova and Polenov [ 101 using Gomori stains. Furthermore, our observations demonstrate that this response was prese n t as early as eight weeks following surgery and persisted for at least 2 8 months. T h e regenerative response in our preparations was substantial. T h e new neurohemal units were reconstituted not only from vessels proliferating within t h e scar, sometimes even within areas of necrotic parenchyma, but also from preexistent vessels that normally d o not contain neurosecretory terminals. These included perforating hypothalamic arterioles, pial vessels, and the vessels of the pars tuberalis of the pituitary gland. Similar innervation of the vessels of the pars tuberalis by magnocellular axons is regularly observed in the proximal stump following division of the pituitary stalk [20,27]. Whether or not the newly formed vessels are structurally similar to those naturally occurring in the median eminence or neuro-
hypophyseal vessels can be determined only by electron microscopy studies. Raisman [26] and others [18, 241 have described in rats the appearance of fenestrated capillaries in the newly formed neural lobe after pituitary stalk section, indicating that the new neurohemal units are similar to the normal. O n e interesting observation in our material was that exuberant regeneration occurred even when the axon was interrupted close to the perikaryon of origin. This observation seems to challenge t h e accepted idea that axonal lesions placed close to the cell body are more likely to result in cell death [15]. Furthermore, both the supraoptic and the paraventricular nucleus showed regenerative ability, but the magnitude of the response depended o n the degree t o which magnocellular elements were preserved. Maintenance of adequate blood supply to the nuclei was also essential to the reconstructive process. Similar observations have been made in animals that have undergone pituitary stalk section [9] or received lesions in other neuronal systems [32]. T w o morphological patterns characterize central nervous system regeneration [21, 301. In one, called regenerative sprouting, the proximal segment of a severed axon sends new sprouts, which in favorable situations may innervate the area that was originally denervated. In the other, designated collateral
Antunes et al: Regeneration of Hypothalamic Magnocellular System 467
sprouting, the denervated sites receive new nerve terminals from normal adjacent axons. We believe o u r observations are m o r e consistent with regenerative sprouting; collateral sprouting, which has now been documented in a number of neuronal systems [ 2 2 , 251, does not seem to apply to o u r observations. W e have shown previously [2] that lesions in the paraventricular nucleus and its tract cause loss of neurosecretory fibers in the zona externa of the median eminence, and in this situation, collateral sprouting does not occur either from adjacent axons of the supraoptic nucleus or from axons of the paraventricular nucleus going t o the infundibular process. A regenerative response of similar magnitude involving aminergic and cholinergic fibers in the rat has been extensively described [G, 17, 30, 331. Some of the morphological features noted in those experiments are similar t o o u r observations: the presence of regenerated nerve fibers in necrotic areas, the terminations around blood vessels, and the presence of positive fibers in areas away from the existing tracts [G, 171. T h e catecholaminergic systems and the magnocellular system have some common features: both are made u p of long, thin, unmyelinated axons which are phylogenetically old, and these qualities seem to favor regeneration of neuronal systems [ 3 3 ] . They are different, however, in that the magnocellular axons synapse mostly, if not totally, with blood vessels. T h e impetus for regenerative o r collateral sprouting remains to be determined. Is it the degenerating axon o r the postsynaptic site? Or is this, as suggested by some [29], just part of a normal organizing process of the adult nervous system? Favorable immunological conditions, such as the lack of a myclin sheath, have been postulated by Berry and Riches [ 4 ] to explain such unique regenerativc capacity. W e do not believe, however, that t h e pituicytes play a significant role, as suggested by Kiernan [ 181, since the interposition of a Silastic barrier following stalk section [ l ] does not preclude regeneration of the proximal stump. N e r v e growth factor seems to increase the degree of regeneration in the aclrenergic systems 171. O n e of the problems that afflicts regeneration in the central nervous system is that growing axons may form "wrong" connections, particularly following collateral sprouting. T h e system therefore loses specificity, and this may limit the functional efficacy of the sprouting axons [lg]. In the magnocellular system, however, specificity is maintained, although we cannot ascertain in o u r preparation how effective the new units were since in all o u r animals a number of neurohemal units were left intact and diabetes insipidus was not observed. Morris and Dyer [23] have demonstrated in the rat that synthesis of ncurose-
468 Annals o f N e u r o l o g y
Vol S
No 5
May lcI79
cretory material continues in paraventricular cells after the axons have been divided by similar techniques. T h e hypothalamic magnocellular system offers particular advantages for morphological and physiological investigations: both nuclei and fiber tracts are easily identifiable for specific immunocytochemical procedures, and the system's functional status may be assessed by radioimmunoassay techniques. T h e study of the response of this system to a variety of lesions, of the temporal development of the response, and of the limiting o r promoting mechanisms may provide important information on the regenerative process in the central nervous system.
We are grateful to D r A. G. Robinson for preparing the rabbir antisera to human estrogen-rtimulated and n i c o r i n ~ - s r i m u l a r e ~ ~ neurophysin; to D r R. D. Utiger for supplying rabbit antiserum r o arginine vasopressin; to Dr P. Czcrnichow for supplying rabbit ;intiserum r n synthetic oxytocin; to Dr K. C. Hsu f o r providing sheep antirabbit globulin serum; to D r L. A. Stcrnberger for giving us the peroxidase-antipcroxidase complexes; and to D r M. Manning for providing synthetic oxytocin and arginine vasopressin.
References I . Antunes JL. Carmel PW, Ferin M , ct al: Section of the pituitary stalk in rhesus monkeys: endocrine funcrion and immunocyrochemical observations. T h e Endocrine Society Manuscript 76, 1977 2. Antuncs JL. Carmel PW. Zimmerman EA: Projections from the paraventricular nucleus to the zona externa of the median eminence of the rhesus monkey: an immunohisrc,chemical study. Brain Res l 3 7 : I L I O . 1977 3. Antunes JL, Zimmerman EA: T h e hypothalamic magnocellular system o f the rhesus monkey. an immunocytochcmical study. J C o m p Ncurol 1 3 1 : s 3')-566, 1978 4 . Rcrry M . Richcs A C : An immunological approach to regeneration in the ccntral nervous system. Br Med Bull 30:135- 140, 1074
5. Billrnstien D
The hypothalamic magnocellular system of the rhesus monkey was studied with specific immunocytochemical techniques in animals that had undergone hypothalamic lesions. The results indicate that this system maintains a regenerative capacity even when its tracts are interrupted within the hypothalamus. New neurohemal units are reconstituted from newly formed vessels within the scar as well as from preexistent blood vessels, such as perforating and pial arterioles, and the vessels of the pars tuberalis of the pituitary gland, which normally do not contain neurosecretory terminals. Antunes JL, Carmel PW, Zimmerman EA, et al: Regeneration of the magnocellular system of the rhesus monkey following hypothalamic lesions. Ann Neurol 51462-469, 1979
It is generally accepted that the central nervous system of the higher vertebrates has a limited ability to regenerate following injury [8, 161. T h e hypothalamic magnocellular neurosecretory system, which includes the supraoptic and paraventricular nuclei and their axons, constitutes an exception to this rule, and in several species section of the pituitary stalk is followed by formation in the proximal stump of a “new” neural lobe [5, 9, 12, 20, 27, 28, 3 11. This phenomenon usually becomes apparent within two to four weeks after operation and depends on the outgrowth of axons of the surviving magnocellular elements, which establish new neurovascular connections. These structures are particularly striking in preparations stained by the Gomori technique. W e have previously investigated the morphological features of the hypothalamic magnocellular neurosecretory system in the rhesus monkey using immunocytochemical techniques and specific antisera to vasopressin, oxytocin, and the associated neurophysin proteins [ 3 ] . W e have observed that the cells containing vasopressin also have so-called nicotine-stimulated neurophysin (NSN), and the cells with oxytocin also contain so-called estrogenstimulated neurophysin IESN). T h e axons of cells of the paraventricular nuclei run mostly laterally and ventrally, and when they reach the optic tract they join the fibers from the supraoptic nuclei and form a compact bundle which passes medially, ventrally, and
posteriorly behind the optic chiasm and courses under the bottom of the third ventricle into the median eminence. Some of the fibers that originate in the paraventricular nucleus terminate around the blood vessels of the zona externa of the median eminence [2]. T h e remaining fibers are arranged in longitudinal bundles that run in the infuridibular stem and terminate on the capillaries of the neural lobe. As demonstrated by immunocytochemical techniques, in intact animals the axons of the magnocellular nuclei d o not make connections with blood vessels in the pia or pars tuberdis, or with perforating hypothalamic arterioles. During investigations on the effects of various hypothalamic lesions o n endocrine function in the rhesus monkey, we found morphological evidence that regeneration of the magnocellular system may occur within the hypothalamus itself. When axons from both the paraventricular and the supraoptic nucleus are divided, the proximal segments form neurovascular connections with newly developed blood vessels within the surgical scar as well as with preexistent blood vessels which normally d o not contain neurosecretory terminals. This paper reports these observations.
From the Departmcnrs of Neurological Surgery and Neurology and the lnbritutc for the Study of Human Reproduction. Columbia University College of Physicians and Surgeons, h e w York. N Y .
Address reprint requests ro Dr Antunes, Department o f Nrurosurgery. Columbia University College of Physicians and Surgeons, 710 W 168th Sr, NKWYork, N Y 10032.
Materials and Methods The brains of 10 adult female rhesus monkeys weighing 3.5 to 4.5 kg were studied. All animals received hypothalamic lesions inflicted with a modified Halasz knife, as describecl
Accepred for publication Oct 17. 1978.
462
0364-5 134/79/050462-08$01.25 @ 1978 by J. Lob0 Antunes
previously [ 141. The purpose was to isolate the medial basal hypothalamus. which includes the ventromedial and arcuate nuclei and the most ventral extension of the supraoptic nuclei. The knife has a bayonet shape with a double-edged steel blade 1 mm wide and 6 mm high with a 3 mm radius of rotation. With the animal under pentobarbital anesthesia and with its head secured in a stereotactic frame, a burr hole was placed at the level of the coronal suture, the superior longitudinal sinus was ligated and divided, and the dura was opened. The knife was introduced vertically in the midline and the tip of the blade placed 15 mm in front and 1 or 2 mm below the external auditory meatus. The knife was then rotated 360 degrees and removed. The animals survived 2 to 28 months, after which they were anesthetized with pentobarbital and the brains fixed by cardiac perfusion with normal saline solution followed by filtered Bouin solution or 109;;;. buffered formalin. T h e calvarium was removed, the dura opened widely, and the brain immersed in the same fixative for a week. T h e hypothalamus and pituitary gland were then removed and fixed for another three to five days. T h e blocks were subsequently washed in running tap water overnight, dehydrated in graded ethanols, replaced with xylene, and embedded in paraffin. The blocks were cut in serial 6 p m coronal sections. Every tenth section was stained with cresyl violet and with hematoxylin and eosin. The immunocytochemical procedures have been described previously [2, 31. Indirect immunoperoxidase techniques using peroxidase-antiperoxidase complexes (PAP) were performed on deparaffinizec{ rehydrated sections, The following primary antisera were used: (1) rabbit antiserum to human ESN and human NSN, used at 1:5,000 and 1:500 dilutions, respectively, for 1 hour at 4°C; antisera to human neurophysins were used because neurophysins of man and rhesus monkey appear to be immunologically identical (see [31); ( 2 ) rabbit antiserum to arginine Vasopressin (7F). used at 1:1,000 for 48 hours at 4°C; and ( 3 ) rabbit antiserum to synthetic oxytocin (69.7). used at 1:250 to 1: 1,000 for 48 hours at 4°C. The secondary antiserum was sheep antirabbit globulin serum, used at 1:100 dilution for 30 minutes at room temperature. PAP was diluted at 1:1,000 for the neurophysin studies and 1:200 for use with oxytocin and vasopressin. Reaction products were formed with 3,3’-diaminobenzidine; some sections were then counterstained with cresyl violet. Controls were obtained by replacing primary antisera with nonimmunc rabbit serum or buffer. Antisera to ESN and N S N were absorbed by addition of 10 pg of purified ESN o r N S N t o 1 ml of anti-ESN (1:5,000) or anti-NSN (1:500) for 24 hours prior to use. Antisera to oxytocin and vasopressin were absorbed by adding 100 ng of synthetic oxytocin and arginine vasopressin, respectively, per 0.1 ml of 1:1,000 dilution.
Results T h e animals survived the surgical procedure without neurological deficit except for 1 monkey that became paraplegic due to injury of the anterior cerebral arteries. T h e results of the endocrine evaluation in
F I R 1 . Discrete gliotic scar containirrg a nrimber of newk formed blood ressels marking the trajeitory o f t h e kniye. Note a nrrnrber of sripranptic cells lateral l o the scar (arrow). IH&E: ~180.)
these animals have been published elsewhere [ 141. None of the animals had permanent or transient diabetes insipidus. The cuts successfully produced hypothalamic disconnections, but bilateral lesions were created in only 5 animals; the other 5 had unilateral disconnections. T h e incomplete sections were due to displacement of the wall of the third ventricle without penetration or injury by the knife on the intact side. The lesions consisted of discrete linear scars that extended from the wall of the third ventricle laterally and inferiorly to the dorsum of the optic tract. In 3 animals they reached the pial surface. T h e scar was made of glial cells and newly formed blood vessels (Fig 1). In 8 animals the nuclear structures medial and lateral to the scar were well preserved except for the paraventricular nucleus, which was invariably damaged by the shaft of the knife. In 2 other animals the knife caused extensive areas of necrosis and cavitation, which included the anterior hypothalamus, the dorsomedial and ventromedial nuclei, and part of the posterior hypothalamus and premammillary region. These lesions were unilateral in 1 monkey and bilateral in the other. They were made of multiple cysts separated by fine, fibrous septa containing newly
Antunes et al: Regeneration of Hypothalamic Magnocellular System
463
formecl branching vessels. These vessels also surrounded the necrotic areas. A number of wellpreserved neurons, many of which belonged t o the paraventricular nucleus, were seen very close to the damaged regions. 1rtrtt~utzoc:ytoibenIiiuiStudies T h e results of the imniunocytocheniical studies were similar for all antisera, indicating that cells containing vasopressinlNSN and the elements containing oxytocinlESN had similar regenerative potential. I n general, the anti-ESN and anti-NSN antisera gave more intense staining. T h e surgical scars interrupted the tracts from the paraventricular and supraoptic nuclei to a variable extent (Fig 2). In these animals, heavy accumulations of immunoreactive granular material were seen around the blood vessels of the scar, and there were also extravascular deposits with large droplets resembling Herring bodies (Fig 3 ) . T h e s e accumulations clearly demarcated the trajectory of the knife. T h e animals with necrotic lesions had extensive deposits of reactive granules surrounding new blood vessels that proliferated around areas of cavitation (Fig 4 A ) . Similar accumulations were also seen around the branching vessels within the septa forming the multiloculated areas (Fig 4 B ) . T h e s e lesions were located within the field of the paravencricularsupraopticoneurohypophyseal tract. Neurosecretory material was also seen around blood vessels in areas not crossed by magnocellular axons, such as in t h e
B
A
464
Annals ot Neurology
Vol 5
No 5
May 1970
subependymal region beneath the floor of the frontal horn of the lateral ventricle (Fig 5 ) . In the 3 animals in which the cut extended ventrally to the pial surface, there were extensive accumulations of reactive material around thc pial vessels and the pars tuberalis of the pituitary gland. I n addition, in several animals positive fibers were seen around the perforating hypothalamic arterics (Fig 6). T h e vascular deposits had a distinctive appearance, with secretory peptides either accumulated in concentric laycrs or impregnating the entire vessel wall. Frequently, beaded axons with abnormally large varicosities could be traced to these vessels, and occasionally a complete “new” neurohemal unit could be identified (Fig 6C). In many instances the interrupted axons and the areas of cystic necrosis were located very close t o magnocellular perikarya, and it did not appear that the magnitude of the regenerative response always depended o n the length of the remaining proximal segment. However, in 1 animal in which the paraventricular nucleus was completely destroyed, thcre was n o accumulation of secretory peptides in the adjacent scar. D u e to the stereotactic coordinates adopted, paraventricular axons were interrupted
B Fig 3. (A)Rear.tii,ef;bers around blood i,essels i n the scar (arrows). ( B JHigher tttu~qtiij5c~tintt oJ‘A.shnwittg headedJ5her.r drotittd blood r,rsrel.i. A lurge Jroplet oJ- ruattir.e n i ~ t e r i a l(Herrittg hodyi (arrow) is uI.ro .serti. , B o t h N S N : A x180. B X720.)
A n r u n e s er ;iI: Regc5tier;ition of Hpporhalarnic Magnoc.ellular Sysrem
465
much m o r e frequently than t h o s e froin t h e supraoptic nucleus. H o w e v e r , in 3 animals in which SUprdOptic ;ixons w e r e divided very close t o t h e perikarya o f origin, reactive deposits a r o u n d t h e newly proliferating blood vessels w e r e also f o u n d . Cell loss within t h e magnocellular nuclei did n o t exceed 20 t o 30"i of t h e total cell population. T h e perikarya of t h e retained magnocellular elem e n t s with apparently transected axons a p p e a r e d t o he slightly smaller, and their processes less n u m e r ous, than normal (Fig 7).T h e nucleus was reduced in size and t h e nucleolus less eviclent. In addition, t h e positive granular material was m o r e densely aggregated t h e n normal. Addition of' the c o r r e s p o n d i n g h o r m o n e antigens to each o f t h e respective h o r m o n e antisera abolished t h e inimunostaining. Replacing t h e antisera with buffer o r normal rabbit s e r u m revealed only t h e peroxidase activity in blood cells, and occasionally in t h e ependymal cells.
Discussion T h e hypothalamic magnocellular system has a particular ability to regenerate following injury. Desclin and Flament-Duran 113 1 r e p o r t e d that posterior pituitary transplanted into t h e hypoth,, iI a m u s c a n be reinnervated by t h e paraventricular-supraopticoneurohypophyseal tract in t h e rat. D e G r o o t [ 1 I ] m a d e electrolytic lesions in t h e cliencephaIon o f t h e rat and f o u n d in t h e damaged areas, often a t considerable distance f r o m t h e paraventricular nucleus, striictures that closely resembled t h e neural lobe; b u t t h e a u t h o r considered this finding to b e a staining artifact. O u r e x p e r i m e n t s s h o w that t h e magnocellular system retains t h e capacity to f o r m
A
B
466 Annals o f Neurology Vol 5 N o 5
May
1979
A
B
F I R 7 . Paraientricufar tixrleris in an animal i n iobirh a iinifateraffeJioti was placed. ( A )Normal side and tBi fesionerl.ride iti /be J-anie .section. (Both ESN: x 18O.i
neurovascular connections, and thus new neurohemal units, even when the axons are interrupted within the hypothalamus. Similar phenomena were recently described in the rat by Danilova and Polenov [ 101 using Gomori stains. Furthermore, our observations demonstrate that this response was prese n t as early as eight weeks following surgery and persisted for at least 2 8 months. T h e regenerative response in our preparations was substantial. T h e new neurohemal units were reconstituted not only from vessels proliferating within t h e scar, sometimes even within areas of necrotic parenchyma, but also from preexistent vessels that normally d o not contain neurosecretory terminals. These included perforating hypothalamic arterioles, pial vessels, and the vessels of the pars tuberalis of the pituitary gland. Similar innervation of the vessels of the pars tuberalis by magnocellular axons is regularly observed in the proximal stump following division of the pituitary stalk [20,27]. Whether or not the newly formed vessels are structurally similar to those naturally occurring in the median eminence or neuro-
hypophyseal vessels can be determined only by electron microscopy studies. Raisman [26] and others [18, 241 have described in rats the appearance of fenestrated capillaries in the newly formed neural lobe after pituitary stalk section, indicating that the new neurohemal units are similar to the normal. O n e interesting observation in our material was that exuberant regeneration occurred even when the axon was interrupted close to the perikaryon of origin. This observation seems to challenge t h e accepted idea that axonal lesions placed close to the cell body are more likely to result in cell death [15]. Furthermore, both the supraoptic and the paraventricular nucleus showed regenerative ability, but the magnitude of the response depended o n the degree t o which magnocellular elements were preserved. Maintenance of adequate blood supply to the nuclei was also essential to the reconstructive process. Similar observations have been made in animals that have undergone pituitary stalk section [9] or received lesions in other neuronal systems [32]. T w o morphological patterns characterize central nervous system regeneration [21, 301. In one, called regenerative sprouting, the proximal segment of a severed axon sends new sprouts, which in favorable situations may innervate the area that was originally denervated. In the other, designated collateral
Antunes et al: Regeneration of Hypothalamic Magnocellular System 467
sprouting, the denervated sites receive new nerve terminals from normal adjacent axons. We believe o u r observations are m o r e consistent with regenerative sprouting; collateral sprouting, which has now been documented in a number of neuronal systems [ 2 2 , 251, does not seem to apply to o u r observations. W e have shown previously [2] that lesions in the paraventricular nucleus and its tract cause loss of neurosecretory fibers in the zona externa of the median eminence, and in this situation, collateral sprouting does not occur either from adjacent axons of the supraoptic nucleus or from axons of the paraventricular nucleus going t o the infundibular process. A regenerative response of similar magnitude involving aminergic and cholinergic fibers in the rat has been extensively described [G, 17, 30, 331. Some of the morphological features noted in those experiments are similar t o o u r observations: the presence of regenerated nerve fibers in necrotic areas, the terminations around blood vessels, and the presence of positive fibers in areas away from the existing tracts [G, 171. T h e catecholaminergic systems and the magnocellular system have some common features: both are made u p of long, thin, unmyelinated axons which are phylogenetically old, and these qualities seem to favor regeneration of neuronal systems [ 3 3 ] . They are different, however, in that the magnocellular axons synapse mostly, if not totally, with blood vessels. T h e impetus for regenerative o r collateral sprouting remains to be determined. Is it the degenerating axon o r the postsynaptic site? Or is this, as suggested by some [29], just part of a normal organizing process of the adult nervous system? Favorable immunological conditions, such as the lack of a myclin sheath, have been postulated by Berry and Riches [ 4 ] to explain such unique regenerativc capacity. W e do not believe, however, that t h e pituicytes play a significant role, as suggested by Kiernan [ 181, since the interposition of a Silastic barrier following stalk section [ l ] does not preclude regeneration of the proximal stump. N e r v e growth factor seems to increase the degree of regeneration in the aclrenergic systems 171. O n e of the problems that afflicts regeneration in the central nervous system is that growing axons may form "wrong" connections, particularly following collateral sprouting. T h e system therefore loses specificity, and this may limit the functional efficacy of the sprouting axons [lg]. In the magnocellular system, however, specificity is maintained, although we cannot ascertain in o u r preparation how effective the new units were since in all o u r animals a number of neurohemal units were left intact and diabetes insipidus was not observed. Morris and Dyer [23] have demonstrated in the rat that synthesis of ncurose-
468 Annals o f N e u r o l o g y
Vol S
No 5
May lcI79
cretory material continues in paraventricular cells after the axons have been divided by similar techniques. T h e hypothalamic magnocellular system offers particular advantages for morphological and physiological investigations: both nuclei and fiber tracts are easily identifiable for specific immunocytochemical procedures, and the system's functional status may be assessed by radioimmunoassay techniques. T h e study of the response of this system to a variety of lesions, of the temporal development of the response, and of the limiting o r promoting mechanisms may provide important information on the regenerative process in the central nervous system.
We are grateful to D r A. G. Robinson for preparing the rabbir antisera to human estrogen-rtimulated and n i c o r i n ~ - s r i m u l a r e ~ ~ neurophysin; to D r R. D. Utiger for supplying rabbit antiserum r o arginine vasopressin; to Dr P. Czcrnichow for supplying rabbit ;intiserum r n synthetic oxytocin; to Dr K. C. Hsu f o r providing sheep antirabbit globulin serum; to D r L. A. Stcrnberger for giving us the peroxidase-antipcroxidase complexes; and to D r M. Manning for providing synthetic oxytocin and arginine vasopressin.
References I . Antunes JL. Carmel PW, Ferin M , ct al: Section of the pituitary stalk in rhesus monkeys: endocrine funcrion and immunocyrochemical observations. T h e Endocrine Society Manuscript 76, 1977 2. Antuncs JL. Carmel PW. Zimmerman EA: Projections from the paraventricular nucleus to the zona externa of the median eminence of the rhesus monkey: an immunohisrc,chemical study. Brain Res l 3 7 : I L I O . 1977 3. Antunes JL, Zimmerman EA: T h e hypothalamic magnocellular system o f the rhesus monkey. an immunocytochcmical study. J C o m p Ncurol 1 3 1 : s 3')-566, 1978 4 . Rcrry M . Richcs A C : An immunological approach to regeneration in the ccntral nervous system. Br Med Bull 30:135- 140, 1074
5. Billrnstien D
