In vivo oximetry of human bulbar conjunctival and episcleral microvasculature using snapshot multispectral imaging.

Multispectral imaging (MSI) is a well-established technique for non-invasive oximetry of retinal blood vessels, which has contributed to the understanding of a variety of retinal conditions, including glaucoma, diabetes, vessel occlusion, and retinal auto-regulation. We report the first study to use snapshot multi-spectral imaging (SMSI) for oximetry of the bulbar conjunctival and episcleral microvasculature in the anterior segment of the eye. We report the oxygen dynamics of the bulbar conjunctival and episcleral microvasculature at normoxia and at acute mild hypoxia conditions. A retinal-fundus camera fitted with a custom Image-Replicating Imaging Spectrometer was used to image the bulbar conjunctival and episcleral microvasculature in ten healthy human subjects at normoxia (21% Fraction of Inspired Oxygen [FiO2]) and acute mild hypoxia (15% FiO2) conditions. Eyelid closure was used to control oxygen diffusion between ambient air and the sclera surface. Four subjects were imaged for 30 seconds immediately following eyelid opening. Vessel diameter and Optical Density Ratio (ODR: a direct proxy for oxygen saturation) of vessels was computed automatically. Oximetry capability was validated using a simple phantom that mimicked the scleral vasculature. Acute mild hypoxia resulted in a decrease in blood oxygen saturation (SO2) (i.e. an increase in ODR) when compared with normoxia in both bulbar conjunctival (p < 0.001) and episcleral vessels (p = 0.03). Average episcleral diameter increased from 78.9 ± 8.7 µm (mean ± standard deviation) at normoxia to 97.6 ± 14.3 µm at hypoxia (p = 0.02). Diameters of bulbar conjunctival vessels showed no significant change from 80.1 ± 7.6 µm at normoxia to 80.6 ± 7.0 µm at hypoxia (p = 0.89). When exposed to ambient air, hypoxic bulbar conjunctival vessels rapidly reoxygenated due to oxygen diffusion from ambient air. Reoxygenation occured in an exponential manner, and SO2 reached normoxia baseline levels. The average ½ time to full reoxygenation was 3.4 ± 1.4 s. As a consequence of oxygen diffusion, bulbar conjunctival vessels will be highly oxygenated (i.e. close to 100% SO2) when exposed to ambient air. Episcleral vessels were not observed to undergo any significant oxygen diffusion, instead behaving similarly to pulse oximetry measurements. This is the first study to the image oxygen dynamics of bulbar conjunctival and episcleral microvasculature, and consequently, the first study to directly observe the rapid reoxygenation of hypoxic bulbar conjunctival vessels when exposed to ambient air. Oximetry of bulbar conjunctival vessels could potentially provide insight into conditions where oxygen dynamics of the microvasculature are not fully understood, such as diabetes, sickle-cell diseases, and dry-eye syndrome. Oximetry in the bulbar conjunctival and episcleral microvasculature could be complimentary or alternative to retinal oximetry.

An outpatient low-intensity locomotor training programme for paediatric chronic incomplete spinal cord injury.

STUDY DESIGN: Single case study. OBJECTIVES: Intensive locomotor training programmes have recently been implemented in paediatric settings for patients with chronic incomplete spinal cord injury. This case study examines whether a lower-intensity locomotor training programme can improve functional ambulation. SETTING: Tertiary care setting in Melbourne, Australia. METHODS: A pretest-post-test design was used for a 17-year-old boy, 16 months after incomplete spinal injury at T6, who was classified as American Spinal Injury Association (ASIA) level C. He participated in two weekly sessions of locomotor training for a period of 6 weeks. Lower Extremity Motor Score (LEMS), Walking Index for Spinal Cord Injury (WISCI II), 6-min walk test (6MWT), 10-m walk test (10MWT), Timed Up and Go (TUG), and the PedsQL were measured before training, immediately after training and 6 weeks after training had ceased. RESULTS: The WISCI II score improved from 6 at baseline to 9 immediately post treatment and this was maintained at follow-up. The PedsQL score was also significantly improved immediately post treatment and at 6 weeks follow-up. The LEMS and 6MWT improved after the intervention also. CONCLUSION: This case study provides evidence of improvements following a less-intensive locomotor training programme in an outpatient setting. Studies with larger samples are required to fully examine the benefits of this programme.

Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons.

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.

Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing.

After damage to the adult mammalian central nervous system (CNS), surviving neurons have limited capacity to regenerate and restore functional connectivity. Conditional genetic deletion of PTEN results in robust CNS axon regrowth, while PTEN repression with short hairpin RNA (shRNA) improves regeneration but to a lesser extent, likely due to suboptimal PTEN mRNA knockdown using this approach. Here we employed the CRISPR/dCas9 system to repress PTEN transcription in neural cells. We targeted the PTEN proximal promoter and 5' untranslated region with dCas9 fused to the repressor protein Krüppel-associated box (KRAB). dCas9-KRAB delivered in a lentiviral vector with one CRISPR guide RNA (gRNA) achieved potent and specific PTEN repression in human cell line models and neural cells derived from human iPSCs, and induced histone (H)3 methylation and deacetylation at the PTEN promoter. The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript, a construct previously used in CNS injury models. The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells, and enhanced neurite outgrowth after nerve growth factor stimulation. PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection.

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP(+) cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

EVALUATION OF INTERFACIAL FLUID DYNAMICAL STRESSES USING THE IMMERSED BOUNDARY METHOD.

The goal of this paper is to examine the evaluation of interfacial stresses using a standard, finite difference based, immersed boundary method (IMBM). This calculation is not trivial for two fundamental reasons. First, the immersed boundary is represented by a localized boundary force which is distributed to the underlying fluid grid by a discretized delta function. Second, this discretized delta function is used to impose a spatially averaged no-slip condition at the immersed boundary. These approximations can cause errors in interpolating stresses near the immersed boundary.To identify suitable methods for evaluating stresses, we investigate three model flow problems at very low Reynolds numbers. We compare the results of the immersed boundary calculations to those achieved by the boundary element method (BEM). The stress on an immersed boundary may be calculated either by direct evaluation of the fluid stress (FS) tensor or, for the stress jump, by direct evaluation of the locally distributed boundary force (wall stress or WS). Our first model problem is Poiseuille channel flow. Using an analytical solution of the immersed boundary formulation in this simple case, we demonstrate that FS calculations should be evaluated at a distance of approximately one grid spacing inward from the immersed boundary. For a curved immersed boundary we present a procedure for selecting representative interfacial fluid stresses using the concepts from the Poiseuille flow test problem. For the final two model problems, steady state flow over a bump in a channel and unsteady peristaltic pumping, we present an 'exclusion filtering' technique for accurately measuring stresses. Using this technique, these studies show that the immersed boundary method can provide reliable approximations to interfacial stresses.

Strain-specific differences in the effects of cyclosporin A and FK506 on the survival and regeneration of axotomized retinal ganglion cells in adult rats.

The immune response can influence neuronal viability and plasticity after injury, effects differing in strains of rats with different susceptibility to autoimmune disease. We assessed the effects of i.p. injections of cyclosporin A (CsA) or FK506 on adult retinal ganglion cell (RGC) survival and axonal regeneration into peripheral nerve (PN) autografted onto the cut optic nerve of rats resistant (Fischer F344) or vulnerable (Lewis) to autoimmune disease. Circulating and tissue CsA and FK506 levels were similar in both strains. Three weeks after autologous PN transplantation the number of viable beta-III tubulin-positive RGCs was significantly greater in CsA- and FK506-treated F344 rats compared with saline-injected controls. RGC survival in Lewis rats was not significantly altered. In F344 rats, retrograde labeling of RGCs revealed that CsA or FK506 treatment significantly increased the number of RGCs that regenerated an axon into a PN autograft; however these agents had no beneficial effect on axonal regeneration in Lewis rats. PN grafts in F344 rats also contained comparatively more pan-neurofilament immunoreactive axons. In both strains, 3 weeks after transplantation CsA or FK506 treatment resulted in increased retinal macrophage numbers, but only in F344 rats was this increase significant. At this time-point PN grafts in both strains contained many macrophages and some T cells. T cell numbers in Lewis rats were significantly greater than in F344 animals. The increased RGC axonal regeneration seen in CsA- or FK506-treated F344 but not Lewis rats shows that modulation of immune responses after neurotrauma has complex and not always predictable outcomes.

Neurotrophic Factors Used to Treat Spinal Cord Injury.

The application of neurotrophic factors as a therapy to improve morphological and behavioral outcomes after experimental spinal cord injury (SCI) has been the focus of many studies. These studies vary markedly in the type of neurotrophic factor that is delivered, the mode of administration, and the location, timing, and duration of the treatment. Generally, the majority of studies have had significant success if neurotrophic factors are applied in or close to the lesion site during the acute or the subacute phase after SCI. Comparatively fewer studies have administered neurotrophic factors in order to directly target the somata of injured neurons. The mode of delivery varies between acute injection of recombinant proteins, subacute or chronic delivery using a variety of strategies including osmotic minipumps, cell-mediated delivery, delivery using polymer release vehicles or supporting bridges of some sort, or the use of gene therapy to modify neurons, glial cells, or precursor/stem cells. In this brief review, we summarize the state of play of many of the therapies using these factors, most of which have been undertaken in rodent models of SCI.

Lengthening and transfer of hamstrings for a flexion deformity of the knee in children with bilateral cerebral palsy: technique and preliminary results.

Between July 2000 and April 2004, 19 patients with bilateral spastic cerebral palsy who required an assistive device to walk had combined lengthening-transfer of the medial hamstrings as part of multilevel surgery. A standardised physical examination, measurement of the Functional Mobility Scale score and video or instrumented gait analysis were performed pre- and post-operatively. Static parameters (popliteal angle, flexion deformity of the knee) and sagittal knee kinematic parameters (knee flexion at initial contact, minimum knee flexion during stance, mean knee flexion during stance) were recorded. The mean length of follow-up was 25 months (14 to 45). Statistically significant improvements in static and dynamic outcome parameters were found, corresponding to improvements in gait and functional mobility as determined by the Functional Mobility Scale. Mild hyperextension of the knee during gait developed in two patients and was controlled by adjustment of their ankle-foot orthosis. Residual flexion deformity > 10 degrees occurred in both knees of one patient and was treated by anterior distal femoral physeal stapling. Two children also showed an improvement of one level in the Gross Motor Function Classification System.

Retinal ganglion cell neurotrophin receptor levels and trophic requirements following target ablation in the neonatal rat.

Superior colliculus (SC) ablation in neonatal rats results in a rapid increase in retinal ganglion cell (RGC) death. This injury-induced death is reduced by exogenous brain-derived neurotrophic factor or neurotrophin-4/5 (NT-4/5), but the protective effect of these molecules is transient, delaying but not preventing neuronal loss. We sought to discover why neurotrophins only temporarily reduce RGC death after target ablation, focusing on changes in neurotrophin receptor expression and possible changes in growth factor dependency. In unlesioned rats, receptor tyrosine kinase B (trkB) immunohistochemistry revealed no change in the number of trkB positive cells in the RGC layer 24 h after intraocular NT-4/5 injection. However, after SC lesions there were significantly less immunoreactive cells and, surprisingly, even fewer immunoreactive cells in NT-4/5 injected eyes. Semi-quantitative confocal analysis of immunofluorescence intensity revealed an increase in trkB staining in the RGC layer in unlesioned rats 24 h after NT-4/5 injection, whereas in SC-lesioned animals exposed to NT-4/5 there was a significant decrease in staining. To determine whether injured neonatal RGCs can switch their trophic requirements, different doses of ciliary neurotrophic factor were given intraocularly, either alone or combined with NT-4/5. We also tested an SC-derived chondroitin sulfate proteoglycan that has been reported to promote neonatal RGC survival. None of these interventions reduced lesion-induced RGC death 24 or 36 h after SC ablation. In summary, we show that developing RGCs do not shift their trophic dependence to other survival factors following injury; rather, the application of neurotrophins causes a down-regulation of the cognate trkB receptor, presumably altering the long-term responsiveness of neonatal RGCs to exogenous neurotrophins. These data highlight the difficulty in promoting long-term neuronal survival when using one-off administration of recombinant growth factors.

A double-blind randomized placebo-controlled study of oral glutamine in the prevention of mucositis in children undergoing hematopoietic stem cell transplantation: a pediatric blood and marrow transplant consortium study.

Severe mucositis is a common cause of morbidity in hematopoietic stem cell transplant (HSCT) recipients. Glutamine has been shown to reduce mucositis in children receiving chemotherapy. Patients were randomized in a double-blind manner to receive glutamine or glycine at a dose of 2 g/m(2)/dose (maximum dose 4 g) twice daily until 28 days post transplant or discharge if sooner. Mucositis was graded by use of a modified Walsh scale. A total of 120 children were evaluable: 57 children received glutamine and 63 received glycine. The mean mucositis score was 3.0+/-0.3 vs 3.9+/-0.4 (P=0.07) in the glutamine and glycine groups, respectively. The glutamine group demonstrated a reduction in mean number of days of intravenous narcotics use (12.1+/-1.5 vs 19.3+/-2.8 in the glycine group, P=0.03) and total parenteral nutrition (17.3+/-1.7 vs 27.3+/-3.6 in glycine group, P=0.01). There was no statistically significant difference in toxicity between the two groups. Glutamine appears to be safe and beneficial in reducing the severity of mucositis. Strong consideration should be given to include oral glutamine supplementation as a routine part of supportive care of SCT patients.

Quantitative analysis of the role of accessory cells in the development of human blood BFU-E-derived erythroid colonies.

A simplified method for the purification of human peripheral blood erythroid progenitor cells (BFU-E) using standard immunological techniques is described. Following removal of platelets, erythrocytes, nylon-wool-adherent cells, and sheep erythrocyte rosette-forming cells (RFC), BFU-E are routinely concentrated tenfold in the null cell fraction. Null cells plated at low density in erythroid cell cultures containing optimal amounts of methylcellulose, erythropoietin, and fetal calf serum did not give rise to spontaneous erythroid colonies. Coculture of null cells with highly purified, autologous RFC at a ratio of 1:25 yielded well-hemoglobinized erythroid colonies which were noticeably smaller than those found in cultures containing unfractionated peripheral blood mononuclear cells. However, further addition of very low numbers of purified adherent cells to null plus RFC dramatically increased the total hemoglobin content as well as the size and number of BFU-E-derived erythroid colonies. Addition of adherent cells alone to null cells had virtually no effect. Under conditions of optimal stimulation by adherent cells and RFC, the number of erythroid bursts was linearly related to null cells plated over an eightfold range. The synergism exhibited between adherent cells and RFC was not restricted by mismatched histocompatibility antigens. This system should be generally useful in quantitating the roles of more highly purified cellular and molecular populations in human erythropoiesis.

Clinically Unsuspected Prion Disease Among Patients With Dementia Diagnoses in an Alzheimer's Disease Database.

BACKGROUND: Brain tissue analysis is necessary to confirm prion diseases. Clinically unsuspected cases may be identified through neuropathologic testing. METHODS: National Alzheimer's Coordinating Center (NACC) Minimum and Neuropathologic Data Set for 1984 to 2005 were reviewed. Eligible patients had dementia, underwent autopsy, had available neuropathologic data, belonged to a currently funded Alzheimer's Disease Center (ADC), and were coded as having an Alzheimer's disease clinical diagnosis or a nonprion disease etiology. For the eligible patients with neuropathology indicating prion disease, further clinical information, collected from the reporting ADC, determined whether prion disease was considered before autopsy. RESULTS: Of 6000 eligible patients in the NACC database, 7 (0.12%) were clinically unsuspected but autopsy-confirmed prion disease cases. CONCLUSION: The proportion of patients with dementia with clinically unrecognized but autopsy-confirmed prion disease was small. Besides confirming clinically suspected cases, neuropathology is useful to identify unsuspected clinically atypical cases of prion disease.

The projection from different visual cortical areas to the rat superior colliculus.

The rat occipital cortex contains a number of morphologically and physiologically distinct visual areas. Retrograde tracing studies have shown that most if not all of these areas project in a topographic fashion to the ipsilateral superior colliculus (SC). In the present study, small amounts of wheat germ agglutinin-conjugated HRP (WGA-HRP) were injected into adult rat occipital cortex to determine how afferents from the different visual cortical areas are distributed within the various layers of the SC. Cytoarchitectonic criteria were used to help establish the location of the WGA-HRP injections in the cortex. As a further aid to identifying the sites of injection, the distribution of retrogradely labelled cells within the thalamus was mapped in each brain. Analysis revealed a surprising range of visuocortical projections to the rat SC, with input to the majority of tectal laminae. Area 17 projected most heavily to the dorsal stratum opticum (SO) and lower half of stratum griseum superficiale (SGS) with lighter label extending up to the collicular surface. Axons and terminals from area 18 formed two horizontal tiers, one in the middle of the stratum griseum intermediale (SGI) and the other at the border between the stratum album intermediale (SAI) and the stratum griseum profundum (SGP). Periodic puffs of label extended between these horizontal tiers, with a periodicity of 300-400 microns. There was some variability in the labelling pattern in the superior colliculus after area 18a injections, perhaps because this cytoarchitectonic area contains multiple representations of the visual field. Generally the projection from the lateromedial, laterointermediate, and laterolateral parts of 18a was heaviest in the lower half of SO and upper regions of SGI. Lighter label extended up into the lower half of SGS. In the SGI it was common to find periodic puffs of terminal label interspersed with areas devoid of innervation. This periodic pattern was particularly noticeable after WGA-HRP injections into anterior 18a. Two horizontal tiers of label were located at the SO/SGI border and SGI/SAI border (the dorsal being the most dense) with patches of label extending between these tiers every 230-250 microns. There did not appear to be a significant corticotectal projection from the posterior part of the lateral extrastriate region.

The development of acetylcholinesterase activity in normal and transplanted superior colliculus in rats.

Histochemical techniques have been used to examine the development of the enzyme acetylcholinesterase (AChE) in normal and transplanted rat superior colliculus (SC). At birth (P0), relatively little AChE activity was found in SC in situ; however, there was a gradual increase in the intensity of AChE staining in the SC over the first 4 postnatal weeks. In the superficial layers, an increase in AChE activity was first seen in rostromedial SC at P6 and was found throughout the upper tectal layers by P10. An increase in AChE activity in the intermediate layers was apparent by P12 and the adult pattern, characterized by periodic bands of AChE staining, was established by P22. In tectal grafts, the development of AChE activity followed a time course similar to that found in normal SC. Mature tectal grafts contained moderate AChE activity with AChE-positive cells scattered throughout the neuropil. There were however, localized, often spherically shaped areas which displayed relatively intense AChE activity. These AChE-dense areas had a characteristic appearance in adjacent sections stained for Nissl or neurofibrils. Significantly, host retinal input, where present, was always restricted to the AChE-dense regions and it seems certain that these areas are homologous to the superficial layers of normal SC. AChE-rich regions were also present, however, in grafts which received no retinal input and in general the pattern of AChE activity in tectal grafts was strikingly similar, irrespective of their location or connections with the host brain. It would appear, therefore, that much of the AChE activity in tectal transplants, and presumably in SC in situ, is intrinsic to that region and not derived from or dependent upon extrinsic innervation.

Time-course and extent of retinal ganglion cell death following ablation of the superior colliculus in neonatal rats.

This study has examined the deleterious effect of superior colliculus (SC) ablation on the viability of identified retinotectally projecting ganglion cells in the neonatal rat retina. The time-course and extent of lesion-induced retinal ganglion cell (rgc) death has been determined and an estimate obtained for the rate of clearance of individual dying neurons. In order to demonstrate the projection of rgcs to the SC and the subsequent death of these same neurons after SC lesions, the fluorescent dye diamidino yellow (DY) was injected into the left SC of anesthetized 2 day old Wistar rats (P2: day of birth = P0). DY retrogradely labels the nuclei of tectally projecting rgcs; if these identified rgcs subsequently die, their DY-labelled nuclei become pyknotic and can be visualized in retinal wholemounts. At P4 the rats were again anesthetized and the injected area, seen as a yellow patch in the SC, was removed by aspiration. Rats were perfused 2 to 336 hours after the lesion and retinal wholemounts of the right eye were prepared. Control rats received only DY injections and were perfused at times corresponding to the lesioned animals. In three sham-operated rats; the injected SC was reexposed at P4 but the tectal tissue was not removed. In each of the 42 rats that were analyzed, about 10% of the retina containing retrogradely labelled rgcs was counted; the number of pyknotic versus normally labelled rgcs was determined and changes in normal cell density were also assessed. Pyknotic rates in control and sham-operated rats were similar (average 0.8%, n = 11). In SC-lesioned rats, the proportion of pyknotic DY-labelled rgcs increased to about 2.5% 4 to 8 hours postlesion (PL); the peak period of death occurred at 23 hours PL (8.0%). The amount of pyknosis decreased thereafter and most dying cells had been eliminated by 50 hours PL. Phagocytosis of dying cells was a common feature of retinae in SC lesioned rats. In the long-term (336 hours) rats, counts of normal DY-labelled rgcs in corresponding regions of control and lesioned rats revealed an average decrease in rgc density of 47.3% after P4 tectal ablation. Calculations suggest a clearance time of about 3 hours for dying neonatal rgcs.

A comparison of postlesion growth of retinotectal and corticotectal axons after superior colliculus transections in neonatal rats.

We examined, in neonatal rats, the postinjury response of two different axonal systems that project to a common target area in the visual system. Transections across the rostral part of the left superior colliculus (SC) were made in 2- or 6-day-old rats (P2, P6). Lesioned animals were randomly selected into short- or long-term groups. The short-term group was used to determine the efficacy of the lesion technique; 2-6 days after transections, right (contralateral) eyes were injected with horseradish peroxidase (HRP). Complete deafferentation of the SC was achieved in 73% of P2 (n = 22) and 53% of P6 (n = 10) short-term animals. In the long-term group (examined 2-7 months after transection), retinotectal and corticotectal projections were assessed in each animal by using [3H]proline and wheat germ agglutin-HRP, respectively. Examination of a series of sagittal sections revealed that the cut had extended across the entire SC in 63% of P2 (n = 19) and 55% of P6 (n = 12) long-term rats. Despite this, retinal and cortical axons were seen in appropriate layers in postlesion SC in all P2 lesioned animals. Cortical projections caudal to the cut were seen in all P6 rats; however, in these animals, the retinal projection was sparse and not always present. Differences in lesion geometry led to consistent differences in the pattern and extent of ingrowth of retinal and cortical axons into postlesion SC neuropil. The two axonal populations also followed different paths as they grew between prelesion and postlesion SC. It is likely that a number of factors influenced the patterns of postlesion growth, including the relative maturity of the axons and the neuropil into which they were growing. There was also, however, clear evidence of competitive interactions between retinal and cortical axons in postlesion SC that consistently led to greater than normal segregation of the two populations and hence restricted their terminal distributions.

Transplantation of tectal tissue in rats. I. Organization of transplants and pattern of distribution of host afferents within them.

We have examined the maturation of tectal tissue transplanted from fetal rats to the midbrain of newborns and have characterized the distribution of host retinal and cortical afferents within the transplants. The transplants develop characteristic internal order and connections which distinguish them from either embryonic cortex or retina placed in the same region. Host retinal afferents project to clearly circumscribed regions, where they synapse mainly on small dendrites or dendritic spines, and only rarely on vesicle-containing profiles. The retinorecipient areas contain few stained axons in neurofibrillar preparations and are almost always located at the surface of the transplant. There is very little overlap in the input from the two eyes into a single transplant even though the projections from each eye may lie adjacent to one another. Cortical afferents spread more broadly in the transplants, but are largely absent from areas of optic termination and from other more deeply located regions with sparse fiber staining properties. The observations suggest that when placed close to its normal location, tectal tissue can develop a number of features characteristic of normal superior colliculus. Appreciation of the internal order of the transplants makes it possible to investigate the cortical and retinal afferent pathways using physiological techniques.

Transplantation of tectal tissue in rats. II. Distribution of host neurons which project to transplants.

Tectal tissue was dissected from fetal rats and transplanted adjacent to the superior colliculus of newborn rats. The recipient animals were then allowed to survive for 6 or more weeks. Subsequent examination revealed that the transplants generally lay over the host inferior colliculus and rostral part of the cerebellum and had substantial fiber connections with the host superior colliculus. To determine which host areas projected to the transplants, horseradish peroxidase (HRP) was injected into the transplants, and the host brain was examined for the presence of retrogradely filled neurons. Labeled cells were found in nearly 50 host areas. Most of these areas are known to project to normal superior colliculus. There was a consistency between one animal and another in the frequency and density of cell label in the various areas. The projection from host cortex (particularly from visual cortical areas) was the densest and most consistent projection. Other areas which commonly projected into the transplants included pretectum, parabigeminal nucleus, superior colliculus, and the brachial region of the inferior colliculus. Sparse and infrequent projections were found from ventral lateral geniculate nucleus, substantia nigra, zona incerta, and catecholaminergic nuclei. No unequivocally labeled retinal ganglion cells were found. The results indicate that the host projection into the transplants is limited to those areas with axons in the vicinity of the host/transplant interconnection. However, the data also suggest that (1) the relative maturity of particular host pathways at the time of transplantation and (2) some form of preferential or absolute affinity expressed between host axons and transplant cells are also factors which influence the pattern of connections formed between host and transplant.