Expression of Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Neurotrophin-3 mRNAs in Human Cortical Xenografts

Trophic factors play an important role in the development of neurons and glia. In order to study the involvement of neurotrophins in human cortical development, human fetal parietal cortical tissue, obtained after early elective abortions, was transplanted to cortical cavities in immunosuppressed rats. Using in situ hybridization it was demonstrated that nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 mRNAs are expressed in developing human cortical xenografts. We conclude that neurotrophins may play a role in human cortical development and rat-derived astroglial cells could be involved in establishing reciprocal “permissive sites”.


INTRODUCTION
To achieve a better understanding of the involvement of trophic factors in human cortical development, studies of the rodent cortex need to be supplemented with studies of the primate cortex. As an alternative to non-human primates, human-torat xenografts provide an interesting possibility /2,5,14,15,25,30,38-41/. The procurement of tissues atter early elective routine abortions has Reprim address: Christian Humpel, Ph.D.

Neurochemistry Unit
Department of Psychiatry Anichstr. 35 A-6020 Innsbruck, Austria permitted studies of human fetal CNS tissue grafted to immunocompromised rodent hosts and of the expression of different human genes and proteins. Growth factors play an important role in survival and differentiation in the CNS and the guiding/ targeting of nerve fiber growth. Nerve growth factor (NGF) and other neurotrophins, brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5, are all present in the brain, the highest levels of NGF, BDNF and NT-3 mRNAs being found in the hippocampus /1,6,12,24,31,42/. Neurotrophins are also expressed in other brain areas, such as cerebral cortex, and may have important functional roles in this brain area, as shown in vitro and in vivo /3,4,10, 13,17,20, 22,29,34,35/. Recently, we demonstrated that human fetal cortical tissue fragments survive grafting to cavities ofthe rat brain/16/. These tissue fragments became integrated with the host brain and formed reciprocal nerve fiber connections /16/. The ingrowth or outgrowth of nerve fibers seemed to occur at specific sites, so called 'ermissive sites", probably guided by trophic interactions /16/. The human grafts significantly expressed angiotensinogen mRNA and protein, indicating a role for this neuropeptide precursor in cortical development /18/. In a recent study we demonstrated BDNF mRNA in developing rat cortex, grafted in oculo to the anterior eye chamber /17/. The aim of the present study was to observe whether the neurotrophin mRNAs (NGF, BDNF, NT-3) are expressed in developing human cortical xenografts. We showed that all three neurotrophins were found in these grafts and rat-derived astroglial cells could be involved in establishing reciprocal connections between graft and host.

MATERIALS AND METHODS
Grafting procedure Grafting was performed as described in detail previously /16,18/. Human fetal parietal cortical fragments were recovered after first trimester (8.5-9.5 weeks) abortions. Women admitted for elective abortions, who had no prior knowledge of this study, were informed of the aims of the study and gave their consent. Anonymity was strictly maintained. The abortions were performed according to hospital routine, with low-pressure vacutun aspiration and with approval by the Regional Ethical Committee of the Karolinska Institute. A cavity in the cortex (bregma: -1 to -2 laterally) of halothane anesthetized female Sprague Dawley rats (B&K Laboratories AB, Sweden, 200-220g, n=5) was made by gentle suction with a modified glass pipette connected to an aspiration pump. The cavity was filled with gelfoam (Spongostan) soaked in Hanks' balanced salt solution (HBSS; Gibco). After 1-2 weeks, the gelfoam was removed under anesthesia to expose the cavity. Human cortical tissue pieces (1-2 mm 3) were placed at the bottom of the cavity and the cavity closed with gelfoam soaked in HBSS. For immtmosuppression all rats received daffy injections of cyclosporine A (10 mg/kg; Sandoz) containing vibramycin (2 mg/kg; Pfizer), starting the day before grafting. The total number of grafts studied was five.
Sections were subsequently rinse,d, washed four times (15 min each) at 54C in lxSSC, cooled to room temperature, dehydrated through 70%, 90% and 99% ethanol and air dried. Sections were dipped in Kodak NTB-2 photo emulsion (diluted 1:1 in water), exposed for 6 (truncated trkB) and 15 weeks (neurotrophins) at -20C, developed, fixed, and lightly counterstained with cresyl violet. Sections were analyzed using light and dark-field microscopy (Nikon Microphot) and photographed (Kodak Tmax-400). Slides were evaluated under the dark field microscope and the signal estimated using a semi-quantitative scale from 0-4.
The following antisense oligonucleotides were used:

RESULTS
All neurotrophin oligonucleotides showed cross reaction with rat and gave the expected hybridization signal in host rat hippocampus ( Fig. 1, upper panel). NGF mRNA was strongly expressed in neurons in and around the host granule and pyramidal cell layer of hippocampus, BDNF mRNA was found in neurons in the granule and pyramidal cell layer of the hippocampal formation and NT-3 mRNA was seen in neurons ofthe granule cell layer of dentate gyms and in CA2 and medial CA1 areas.
All three neurotrophins were expressed in the developing human cortical xenografts (  (Table 1). A strong patchy expression of BDNF mRNA was seen in the human cortical grafts, qe signal for BDNF mRNA was highest in the youngest graft (16.5 weeks), slightly decreased thereafter, reaching a moderate expression in the older grafts (Table 1).  mRNA was Ibund in the gratis in a patchy pattern. The signal for NT-3 mRNA peaked at week 25 (Table l).
In situ hybridization for rat tnmcated trkB receptor mRNA showed a strong signal over the host cortex (Fig. 2a,b). The gratis partly revealed higher background but did not show a specific VOLUME 5, NO. 4, 1995   High levels of expression of rat truncated trkB receptor mRNA are found in the rat host cortex (ctx), while the human transplants (tp) have a somewhat higher background but are negative (a, b). Somewhat stronger signals are found close to the graft-host interface (a, b). The box in Fig. a represents the area depicted at higher magnification in Fig. b. In a few areas a strong signal is found representing "permissive sites" (large arrows, c, d). Bright field microscopy reveals specific localization (small arrows) of silver grains over cresyl violet counterstained cells (d). Bar 240 tm (a), 100 (b), 150 tm (c), 50 tm (d). signal ( Fig. 2a-d). Often the hybridization signal was increased at the graft-host interface (Fig. 2b).
In a few cases a strong signal was found in ratderived cells, possibly in areas where nerve fibers entered the transplant (Fig. 2c,d).

DISCUSSION
This study is an extension of our previous studies in which human-to-rat cortical xenografts /16,18/ and rat cortex in oculo grafts /17/ were characterized. Briefly, the majority of the grafts survived, became integrated with the rat host cortex and formed reciprocal nerve fiber connections. Specific 'permissive sites" seem to be involved in this nerve fiber growth from graft to host and from host to graft. The present study demonstrates that NGF, BDNF and NT-3 mRNAs were expressed in the developing human cortical xenografis.
NGF, BDNF and NT-3 cross reacted with rat and gave the expected pattern of expression in the rat hippocampus/9,12,31/. The probe for truncated trkB receptor gave strong signals all over the rat host brain, especially in rat cortex/19/. The positive cells in the grafts expressing the neurotrophins and their receptors were identified using bright field microscopy, showing the silver grains to be located over cresyl violet counterstained cells.
Several studies have indicated a role for neurotrophins in cortical development in the rat. Using in situ hybridization, BNDF mRNA was found in several cortical areas (frontal, parietotemporal, cingulate and piriform) in the developing rat from 2 weeks of age and thereafter/10/and in prefrontal cortex of fetal and adult monkeys/20/. Exogenous NGF affected the development of graRed cortex cerebri /8/ and BDNF, and much less potently NT-3, regulated neuropeptide expression in cultured cerebral cortical neurons/29/. Castrrn et al. /3/ demonstrated that physiological stimuli (visual input changes) can regulate BDNF and trkB mRNAs in the visual cortex. Neurotrophins also seem to play an important role in glutamatemediated excitotoxicity in cortical neurons/22,33, 34/. Recently, we reported/17/that BDNF mRNA as well as trkB receptor mRNAs were expressed in rat cortex cerebri grafted to the anterior chamber of the eye. BDNF mRNA expression was increased compared to adult cortices in single grafts and downregulated to near control levels by a compensating second graft, indicating trophic interactions/17/. The present study extends these reports, and describes that all three analyzed neurotrophins, NGF, BDNF and NT-3, are expressed in the human cortical xenograRs, likely playing important roles during development. Indeed, a recent report showed that BDNF is required for activity-dependent survival of cortical neurons/13/. Since it was not easy to obtain several grafts ofthe same age in this study, the different age data need to be carefully interpreted. It might be possible that the different levels of mRNA expression do not represent normal developmentally regulated mRNA expression, but are merely a reflection of intra-transplantation or intra-donor variability.
Earlier studies of similar graRs suggested that most cells in our human grafts were immature/18/. Recently, we reported that the mRNA for a neuropeptide precursor, human angiotensinogen, was expressed mainly in immature protoplasmatic astrocytes in human cortical grafts/18/. As we were not able to use non-radioactive in situ hybridization combined with glial immunomarkers, we cannot determine whether the neurotrophins are expressed in immature neurons and/or glial cells as well.
However, since the localization of neurotrophins in glial cells has not been demonstrated by in situ hybridization in vivo, we suggest that the neurotrophin mRNAs are expressed in immature neurons in the human cortical grafts. This is in agreement with Miranda et al. /28/, who reported that NGF and BDNF and more rarely NT-3 mRNAs were expressed in the developing rat cerebral cortex.
The neurotrophins bind with high affinity to receptors encoded by members of the trk family of proto-oncogenes/6,7,32/. The trk-related gene trkB encodes a high-affinity signal transducing component, having high affinity for both BDNF and NT-3 /36,37/. The full-length trkB contains a tyrosine kinase domain, while truncated trkB lacks an intracellular signal-transducing domain /21,27/. In the CNS, the full-length trkB mRNA is predominantly expressed in neurons, while expression of truncated trkB is predominantly found in glial cells/11,21,44/. Miranda et al. /28/ recently demon-strated that the trkB receptor mRNA is expressed in the developing rat cerebral cortex, and is coexpressed with different neurotrophins. Kumar et al. /23/reported that trkB is present in astrocytes and upregulated by NGF, suggesting a role in normal glial function. In our study, truncated trkB mRNA expression was otten very strong in areas at the graft-host interface to the cortical grafts indicating that rat astroglial processes are involved. We have also observed a similar phenomenon in intraoculo rat cortical transplants/17/. The present data indicate that glial processes and the high affinity trkB receptor might be involved in neuronal and glial communication at the graft-host border.
"Permissive sites" seem to be responsible for establishing reciprocal newe fiber connections.
Several cases of such '])ermissive sites" were found in our human cortical gratts and nerve fibers labeled with human-specific neurofilament antibody entered such sites (for details see/16,18/). We report now that these 'permissive sites" appear to contain ratderived cells expressing the truncated trkB receptor mRNA. These cells seem to form an entry for the ingrowth (or outgrowth) or nerve fibers, starting from a specific point at the host-graft interface. We suggest that these cells are rat astrocytes, because truncated trkB is predominantly found in glial cells and neu.rons, while the expression of full length trkB is restricted to neurons. At the 'permissive sites", it might be possible that astroglial processes are necessary for guiding/targeting nerve fiber growth and that neurotrophins could play a role in this process. lis study shows that mRNAs for NGF, BDNF and NT-3 are expressed in developing human cortical gratis. We conclude that neurotrophins may play a role during development and nerve fiber growth in the human cortex, and rat-derived astroglia could be involved in establishing reciprocal connections.