Albino Rat Eyes Research Articles

Novel Needle for Intravitreal Drug Delivery: Comparative Study of Needle Tip Aspirates, Injection Stream and Penetration Forces.

PurposeTo study the efficacy of a novel needle for intravitreal injection (IVI) in comparison to the conventional needle under experimental conditions.MethodsThe newly designed 30-gauge (G) needle (NDN) (EP 18158 542.3, patent pending) with occluded outer orifice and a side port for drug delivery was compared to the conventional standard hypodermic 30 G needle for IVI (SHN). An animal study to obtain needle tip aspirates was performed on 10 albino rat eyes. During IVIs, cellular content, which was cut by the needle tip, was aspirated. Cellular material was studied in regard to cell types and their quantity. The injection stream was studied using trypan blue dye in vitro and pig cadaver eyes. The penetration force was tested on polyurethane Testing Foil Strips PU 04 (Melab, Leonberg, Germany) by applying a velocity of 100 mm/min. The results were analyzed using descriptive statistics, correlation matrices and t-test methods with p<0.05 as statistically significant.ResultsCytological analysis of the needle aspirates showed the presence of cellular content in each case. The amount of conjunctival, ciliary body epithelial cells and granulated basophilic protein sediments (sign of cellular damage) in the case of the NDN tips was significantly lower compared to the SHN. The average penetration force of the NDN was 0.791 N, and in the case of the SHN was 0.566 N. The injection stream study revealed a difference in the initial injection phase between the two needle types, although the diffuse filling of the vitreous area which surrounded the needle tip appeared to be similar.DiscussionThe NDN demonstrated superior performance with regard to a significantly reduced number of cells being captured by the needle tip. Delivery of the injected fluid into the vitreous cavity was comparable. In order to investigate superior properties of the NDN needle design, further studies with improved prototypes would be necessary.

Efficacy of topotecan nanoparticles for intravitreal chemotherapy of retinoblastoma

Retinoblastoma (Rb) is the most common intraocular malignancy in children that accounts for approximately 4% of all pediatric malignancies. Since chemotherapy is a widely practiced treatment for Rb, there is a growing interest in developing new and effective drugs to overcome systemic and local side effects of chemotherapy to improve the quality of life and increase the chances of survival. This study sought to fabricate thiolated chitosan nanoparticles containing topotecan (TPH-TCs-NPs) with a view of enhancing drug loading and release control. This research was also designed to assess the ability of TPH-TCs-NPs to improve cell association, increase treatment efficacy in retinoblastoma cells and xenograft-rat-model of retinoblastoma, and overcome current topotecan hydrochloride (TPH) intravitreal administration challenges, including stability loss and poor cellular uptake. Modified ionic gelation method was optimized to fabricate TPH-TCs-NPs and TPH-TMC-NPs (N-trimethyl chitosan nanoparticles containing TPH). We characterized the NPs and quantified topotecan loading and release against a free TPH standard. The efficacy of TPH-NPs was quantified in human retinoblastoma cells (Y79) by XTT and flow cytometry measurement. In addition, Y79 cells were injected intravitreally in both eyes of immunodeficient wistar albino rats to create a xenograft-rat-model to compare the antitumor effectiveness of TPH-NPs and TPH by intravitreal administration. TPH-NPs complexation was confirmed by EDX, FTIR, and DSC techniques. TPH-TCs-NPs and TPH-TMC-NPs had high encapsulation efficiency (85.23 ± 2 and 73.34 ± 2% respectively). TPH-TCs-NPs showed a mean diameter, polidispersity index, and zeta potential of 25±2 nm, 0.21 ± 0.03 and +12 ± 2 mV, respectively. As a function of dose, TCs and TMC NPs were more efficacious than free topotecan (IC50s 53.17 and 85.88 nM, relative to 138.30 nM respectively, P = 0.012). Kruskal-Wallis test showed a statistically significant difference between the groups. Additionally, a significant difference between the tumor control and TPH-TCs-NPs treated group in xenograft-rat-model ( Range of P-value: 0.026 to 0.035) was shown by Bonferroni post hoc test. The current investigation demonstrated enhanced efficacy and association of TPH-TCs-NPs relative to free TPH in retinoblastoma cells and tumor in vitro and in vivo.

In vivo blind-deconvolution photoacoustic ophthalmoscopy with total variation regularization.

Photoacoustic ophthalmoscopy (PAOM) is capable of noninvasively imaging anatomic and functional information of the retina in living rodents. However, the strong ocular aberration in rodent eyes and limited ultrasonic detection sensitivity affect PAOM's spatial resolution and signal-to-noise ratio (SNR) in in vivo eyes. In this work, we report a computational approach to combine blind deconvolution (BD) algorithm with a regularizing constraint based on total variation (BDTV) for PAOM imaging restoration. We tested the algorithm in retinal and choroidal microvascular images in albino rat eyes. The algorithm improved PAOM's lateral resolution by around 2-fold. Moreover, it enabled the improvement in imaging SNR for both major vessels and capillaries, and realized the well-preserved blood vessels' edges simultaneously, which surpasses conventional Richardson-Lucy BD algorithm. The reported results indicate that the BDTV algorithm potentially facilitate PAOM in extracting retinal pathophysiological information by enhancing in vivo imaging quality without physically modifying PAOM's optical configuration.

Ocular fundus pulsations within the posterior rat eye: Chorioscleral motion and response to elevated intraocular pressure

A multi-functional optical coherence tomography (OCT) approach is presented to determine ocular fundus pulsations as an axial displacement between the retina and the chorioscleral complex in the albino rat eye. By combining optical coherence elastography and OCT angiography (OCTA), we measure subtle deformations in the nanometer range within the eye and simultaneously map retinal and choroidal perfusion. The conventional OCT reflectivity contrast serves as a backbone to segment the retina and to define several slabs which are subsequently used for quantitative ocular pulsation measurements as well as for a qualitative exploration of the multi-functional OCT image data. The proposed concept is applied in healthy albino rats as well as in rats under acute elevation of the intraocular pressure (IOP). The evaluation of this experiment revealed an increased pulsatility and deformation between the retinal and chorioscleral complex while increasing the IOP level from 15 mmHg to 65 mmHg. At IOP levels exceeding 65 mmHg, the pulsatility decreased significantly and retinal as well as choroidal perfusion vanished in OCTA. Furthermore, the evaluation of the multi-parametric experiment revealed a spatial correlation between fundus pulsatility and choroidal blood flow. This indicates that the assessed pulsatility may be a valuable parameter describing the choroidal perfusion.

MicroRNA regulation in an animal model of acute ocular hypertension.

PurposeTo analyse miRNA regulation in a rat model of acute ocular hypertension (AOH).MethodsAcute ocular hypertension (AOH) was induced in the left eye of adult albino rats by inserting a cannula connected with a saline container into the anterior chamber. The contralateral eye served as a control. Seven days later, animals were killed. Retinas were used either for quantitative analysis of retinal ganglion cells (RGCs) and microglial cells or for miRNA array hybridization, qRT‐PCR and Western blotting.ResultsAnatomically, AOH caused axonal degeneration, a significant loss of RGCs and a significant increase in microglial cells in the ganglion cell layer. The miRNAs microarray analysis revealed 31 differentially expressed miRNAs in the AOH versus control group, and the regulation of 12 selected microRNAs was further confirmed by qRT‐PCR. Bioinformatic analysis indicates that several signalling pathways are putatively regulated by the validated miRNAs. Of particular interest was the inflammatory pathway signalled by mitogen‐activated protein kinases (MAPKs). In agreement with the in silico analysis, p38 MAP kinase, tumour necrosis factor‐alpha (TNF‐α) and iNOS proteins were significantly upregulated in the AOH retinas.ConclusionsAcute IOP elevation led to changes in the expression of miRNAs, whose target genes were associated with the regulation of microglia‐mediated neuroinflammation or neural apoptosis. Addressing miRNAs in the process of retinal ischaemia and optic nerve damage in association with high IOP elevation may open new avenues in preventing retinal ganglion cell apoptosis and may serve as target for future therapeutic regimen in acute ocular hypertension and retinal ischaemic conditions.

Melanin Pigmentation in Rat Eyes: In Vivo Imaging by Polarization-Sensitive Optical Coherence Tomography and Comparison to Histology.

The purpose of this study was to demonstrate polarization-sensitive optical coherence tomography (PS-OCT) for imaging pigmented structures in the posterior eye segments of albino and pigmented rats and to correlate depolarization contrast of the retinal pigment epithelium (RPE) and choroid in in vivo PS-OCT to melanin pigmentation detected in postmortem histologic serial sections. In vivo three-dimensional PS-OCT imaging was performed in adult albino and pigmented rat eyes at 70-kHz A-line rate. Degree of polarization uniformity (DOPU) fundus maps and radial DOPU profiles were generated. Postmortem histomorphologic analysis was performed in order to investigate melanin pigmentation of the RPE and choroid. Fundus pigmentation maps were extracted from histologic serial sections. Pigmentation profiles were correlated to DOPU profiles of the same eyes. Strong depolarization was found in the RPE/choroid complex of pigmented rats, whereas the same structures exhibited uniform polarization in albino rats. The difference between the depolarization characteristics between albino and pigmented animals was statistically significant. In the fundus pigmentation maps, optical pigment density was zero in albino rat eyes. In pigmented rat eyes, a strong negative correlation between optical pigment density and DOPU was observed. This in vivo and ex vivo investigation of posterior rat eyes indicates that melanin is the cause of depolarization in retinal PS-OCT images. It further demonstrates that melanin pigmentation in the RPE and choroid can be quantified via depolarization imaging and therefore suggests that PS-OCT is a useful tool for the noninvasive quantitative assessment of pigmentary changes in vision-threatening diseases such as age-related macular degeneration.

Investigating the influence of chromatic aberration and optical illumination bandwidth on fundus imaging in rats.

Abstract. Rodent models are indispensable in studying various retinal diseases. Noninvasive, high-resolution retinal imaging of rodent models is highly desired for longitudinally investigating the pathogenesis and therapeutic strategies. However, due to severe aberrations, the retinal image quality in rodents can be much worse than that in humans. We numerically and experimentally investigated the influence of chromatic aberration and optical illumination bandwidth on retinal imaging. We confirmed that the rat retinal image quality decreased with increasing illumination bandwidth. We achieved the retinal image resolution of 10 μm using a 19 nm illumination bandwidth centered at 580 nm in a home-built fundus camera. Furthermore, we observed higher chromatic aberration in albino rat eyes than in pigmented rat eyes. This study provides a design guide for high-resolution fundus camera for rodents. Our method is also beneficial to dispersion compensation in multiwavelength retinal imaging applications.

Fundus Camera Guided Photoacoustic Ophthalmoscopy

Purpose: To demonstrate the feasibility of fundus camera guided photoacoustic ophthalmoscopy (PAOM) system and its multimodal imaging capabilities.Methods: We integrated PAOM and a fundus camera consisting of a white-light illuminator and a high-sensitivity, high-speed CCD. The fundus camera captures both retinal anatomy and PAOM illumination at the same time to provide a real-time feedback when we position the PAOM illuminating light. We applied the integrated system to image rat eyes in vivo and used full-spectrum, visible (VIS), and near infrared (NIR) illuminations in fundus photography.Results: Both albino and pigmented rat eyes were imaged in vivo. During alignment, different trajectories of PAOM laser scanning were successfully visualized by the fundus camera, which reduced the PAOM alignment time from several minutes to 30 s. In albino eyes, in addition to retinal vessels, main choroidal vessels were observed using VIS-illumination, which is similar to PAOM images. In pigmented eyes, the radial striations of retinal nerve fiber layer were visualized by fundus photography using full-spectrum illumination; meanwhile, PAOM imaged both retinal vessels and the retinal pigmented epithelium melanin distribution.Conclusions: The results demonstrated that PAOM can be well-integrated with fundus camera without affecting its functionality. The fundus camera guidance is faster and easier comparing with our previous work. The integrated system also set the stage for the next-step verification between oximetry methods based on PAOM and fundus photography.

Monitoring the effect of mild ischemia with a built-in light-emitting diode contact lens electrode and a low-cost custom-made apparatus

Electroretinography (ERG) is widely used in clinical work and research to assess the retinal function. We evaluated an easy to build ERG setup adapted for small animals comprising two contact lens electrodes with a built-in light-emitting diode and a custom-made amplification system. The system's sensitivity was tested by monitoring ERG in albino rat eyes subjected to mild ischemia. Flash ERG was recorded by two contact lens electrodes positioned on the rat's corneas and used alternately as test or reference. The a- and b-wave amplitudes, a-wave latency, b-wave implicit time and oscillatory potentials (OPs) were analyzed. Ischemia was achieved by elevating the intraocular pressure in the eye's anterior chamber. ERG was recorded on post-ischemia (PI) days −1, 1, 3 and 7. Morphological changes were analyzed on hematoxylin/eosin stained 5 µm sections of control 7d PI retinas. In control eyes, ERG exhibited a pattern similar to a standard recording. Retinas subjected to mild ischemia preserved ordered layered morphology, exhibiting approximately 30% loss of ganglion cells and no changes in gross morphology. By day 3 PI, ischemia caused an increase in the a-wave amplitude (from 34.9 ± 2.7 to 45.4 ± 4.3 µV), a decrease in the b-wave amplitude (from 248 ± 13 to 162 ± 8 µV), an increase in a-wave latency (from 11.1 ± 0.3 to 17.3 ± 1.4 ms) and b-wave implicit time (from 81.0 ± 1.6 to 90.0 ± 2.5 ms), and attenuation of OPs. The described setup proved sensitive and reliable for evaluating subtle changes in the retinal function in small animals.

Near-infrared light photoacoustic ophthalmoscopy.

We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.

New antioxidant SkQ1 is an effective protector of rat eye retinal pigment epithelium and choroid under conditions of long-term organotypic cultivation

Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.

Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform

Photoacoustic ophthalmoscopy (PAOM) is a newly developed retinal imaging technology that holds promise for both fundamental investigation and clinical diagnosis of several blinding diseases. Hence, integrating PAOM with other existing ophthalmic imaging modalities is important to identify and verify the strengths of PAOM compared with the established technologies and to provide the foundation for more comprehensive multimodal imaging. To this end, we developed a retinal imaging platform integrating PAOM with scanning laser ophthalmoscopy (SLO), spectral-domain optical coherence tomography (SD-OCT), and fluorescein angiography (FA). In the system, all the imaging modalities shared the same optical scanning and delivery mechanisms, which enabled registered retinal imaging from all the modalities. High-resolution PAOM, SD-OCT, SLO, and FA images were acquired in both albino and pigmented rat eyes. The reported in vivo results demonstrate the capability of the integrated system to provide comprehensive anatomic imaging based on multiple optical contrasts.

Image chorioretinal vasculature in albino rats using photoacoustic ophthalmoscopy

We imaged the microvascular network in both the retina and the choroid in an albino rat eye using photoacoustic ophthalmoscopy guided by optical coherence tomography. Relying on optical absorption and ultrasonic detection, photoacoustic ophthalmoscopy can image both retinal and choroidal vessel networks with high contrast.

Age-Associated Changes in the Cornea, Lens and Retina of the Albino Rat Eye: A Histological and Immuno-Histochemical Study

Background: Age-related changes occur throughout the body including vision. Aging has various effects on the visual system affecting both external and internal portions of the eye. Age-related visual deterioration may be due to both neural and optical factors. Aim of the Work: To study the normal histological changes of aging on the cornea, lens and retina of albino rat eye. Materials and Methods: In this study twenty-four male albino rats were used. They were divided into Two groups: Group (1) young-aged group (3-6 months) and Group (2) old-aged Group (22-26 months). All animals were sacrificed and their eye balls were extracted and prepared for light microscopic examination. Immuno-histochemical stain was also added to detect apoptosis. Results: It was revealed that aging induced various histological changes in the eye of albino rat. The cornea showed irregular surface with atrophy of the epithelial cells and wide separation of corneal lamellae with loss of keratocytes. The lens subcapsular epithelial cells appeared few and disarranged. The cortical lens fibers lost their nuclei. The retina showed diminished retinal thickness, damage of some ganglion cells and retinal pigmented epithelial cells. The outer nuclear layer revealed marked diminution of its thickness with small dark nuclei. Rods and cones showed decreased density. The Bruch's membrane appeared illdefined and irregular with some adherent deposits and blood leakage in the choriocapillaries. The immunohistochemical stain revealed appearance of some apoptotic cells in the cornea, lens subcapsular epithelial cells and ganglion cell layer as well as pigment epithelial cells of the retina in old-aged rats. Conclusion:Normal aging is associated with some histological and immuno-histochemical changes that could explain the visual impairment observed in elderly

Photoreceptor function of retinal transplants implicated by light-dark shift of S-antigen and rod transducin

The aim was to demonstrate functional properties of transplanted histologically normal photoreceptors. Subretinal intact-sheet transplants of fetal E17–E20 rat retinas to light-damaged albino rat eyes were fixed in light or dark, 2 to 42 weeks after transplantation, and stained immunohistochemically for certain phototransduction proteins. In light adapted transplants, transducin was predominantly found in inner segments of parallel-organized photoreceptors. Transducin shifted to the outer segments with dark-adaptation. S-antigen distribution was opposite to transducin. Rhodopsin distribution did not change. The shift of signal transduction proteins correlated to the light conditions indicates that normal phototransduction processes were established in photoreceptors of transplanted retinal sheets.

2,5-Hexanedione concentrations and morphological changes within the eye of albino rat

Exposure to 2,5-hexanedione (2,5-HD), a major n-hexane metabolite, can cause loss of photoreceptor cells, particularly when combined with light energy. The aims of this study were to document the levels of 2,5-HD reached in relation to time in retina, aqueous humor, and serum of the Sprague-Dawley albino rat after: (1) a single oral administration of 2,5-HD (0.04 g/ kg body wt.) by tube feeding; and (2) after subchronic oral administration of 2,5-HD. In addition, morphometric analysis of the retina was carried out to evaluate cell loss at the end of administration and after various periods of recovery. The 2,5-HD concentration in retinal tissue, aqueous humor, and serum reached a peak within 1 h after exposure to a single dose of 2,5-HD. Twenty four hours after the exposure, only a minor amount of 2,5-HD could be detected in the retina and aqueous humor. When 2,5-HD was administered subchronically (0.04 g/kg body wt. per day, for 35 days) no loss of photoreceptor cells was seen immediately after the end of exposure or at the end of a 4-week recovery period. Rats exposed to 0.60 g/kg body wt. per day for 11 days showed no significant loss of photoreceptor cells immediately after the end of exposure but there was a substantial loss of photoreceptor cells after 2 and 4 weeks of recovery. The results demonstrate that 2,5-HD reaches the aqueous humor and retina, and penetrates blood-aqueous humor/retina barriers after oral administration. Moreover, retinal degeneration seen in the animals may be directly caused by 2,5-HD and these changes are dose dependent.

Lipofuscin in the Retina: Quantitative Assay for an Unprecedented Autofluorescent Compound (Pyridinium Bis-Retinoid, A2-E) of Ocular Age Pigment

The pyridinium bis-retinoid, A2-E, has been discovered as one of the major autofluorescent components of retinal pigment epithelial lipofuscin. Due to its chemical characteristics, A2-E may contribute to cellular and molecular changes leading to age-related macular degeneration. Because A2-E is the first lipofuscin component that has been identified, purified, and its structure analysed, it represents an important marker molecule for studying lipofuscin formation under various conditions. In order to investigate the role of A2-E in ageing processes of the retinal pigment epithelium, we developed an HPLC assay for this compound using single wavelength UV-absorbance detection with continuous light emission. Standard A2-E was synthetized and purified by sequential TLC. In our assay, A2-E can be detected in amounts lower than 10 pmol. The assay has been applied to quantitative determination of A2-E amounts in albino rat eyes of different age groups. Our results demonstrate that there is a marked increase of A2-E levels in older animals. The method described is the first to allow quantification of this unusual retinoid from small amounts of biological samples.

A sensitive ocular perfusion apparatus measuring outflow facility

PURPOSE. To build an improved apparatus for measuring outflow facility by one- and two-level, constant pressure perfusion. METHODS. A computer-controlled and -monitored tower adjusts the height of a fluid reservoir by a stepper motor every 5 min, to keep intraocular pressure (IOP) constant at one or two desired pressure levels (e.g. 2.5 and 12.0 mm Hg above the spontaneous IOP). The fluid flow rate is calculated by the equation Q = (Pres 2 IOP)/R, where Pres is the pressure of the reservoir as calculated by the term "rgh," h being the height of the reservoir, measured by the rotation of stepper motor, g, the gravity constant, and r, the fluid density. IOP is measured by the transducer, and R is the resistance of 180 mm tubing. Outflow facility is calculated by ?Q/?P. ?Q is the difference between flow rate at the two pressure levels, and ?P is the intraocular pressure difference. Perfusion was performed on live albino rat eyes by the two-level constant pressure technique, and on artificial steel eyes at a single constant pressure perfusion (20 mm Hg). RESULTS. Outflow facility of albino rat eyes and artificial steel eyes perfused for 40 and 30 min, respectively was 0.0507 ± 0.0039 µl/min/mm Hg and 0.3546 ± 0.0034 µl/min/mm Hg (mean ± SD), respectively. CONCLUSIONS. Our perfusion apparatus is highly stable, sensitive, and capable of measuring subtle changes in outflow facility in vivo and in vitro.

Immunolocalization of calcitriol receptor, plasma membrane calcium pump and calbindin-D28k in the cornea and ciliary body of the rat eye.

Epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump (PMCA) and the 28-kD vitamin-D-dependent Ca-binding protein (calbindin-D28k) were detected in sections of the albino rat eye using light microscopy and immunohistochemistry of paraffin-embedded tissues. Calcitriol receptor and calbindin-D28k epitopes were detected in both the inner and outer layers of the ciliary body epithelium. PMCA was present in both epithelial cell layers of the ciliary body but was most prominent in the inner layer. Corneal epithelium and endothelium also contained epitopes for calcitriol receptor, PMCA and calbindin-D28k. These Ca-regulatory proteins may play a role in the cellular physiology of the albino rat eye by maintaining appropriate intracellular and aqueous humor Ca concentrations.

Spinal cord-skeletal muscle cografts: trophic and functional interactions

Skeletal muscle from embryonic day 20 (E20) was combined with E15 rat spinal cord in the anterior chamber of the eye of adult albino rats. The two grafts were either transplanted concomitantly or sequentially, in which case muscle tissue was added 4 months after the spinal cord. Control groups received a single graft of either spinal cord or skeletal muscle. Survival and intraocular growth were observed through the cornea. After maturation in oculo, the double grafts were examined immunohistologically utilizing antisera to neurofilament (NF) and acetylcholinesterase (AChE). The grafts were also evaluated using electrical stimulation to determine functional connectivity. The spinal cord and skeletal muscle grafts were found to exert reciprocal tropic effects on each other, evidenced as a larger muscle mass in skeletal muscle grafts allowed to develop in the presence of spinal cord tissue, and a larger volume of spinal cord grafts allowed to develop together with a skeletal muscle graft, respectively. Immunohistochemistry revealed NF-positive nerve fibers leaving the spinal cord graft and entering the muscle tissue. AChE-positive endplates developed in the muscle grafts. Electrical stimulation of the spinal cord part of double-graft combinations generally elicited contractile responses in specific areas of the muscle cograft. These results demonstrate both structural and functional connections between grafts of spinal cord and skeletal muscle tissue in vivo. The fact that such connections were also established between a mature (adult) spinal cord graft and fetal skeletal muścle tissue suggests that some α-motoneurons are able to survive for many months in the intraocular grafts without an appropriate target, and that they are able to subsequently innervate skeletal muscle targets.