Functional and molecular 3D mapping of angiosarcoma tumor using non-invasive laser speckle, hyperspectral, and photoacoustic imaging.

PURPOSE: The gold standard for skin cancer diagnosis is surgical excisional biopsy and histopathological examination. Several non-invasive diagnostic techniques exist, although they have not yet translated into clinical use. This is a proof-of-concept study to assess the possibility of imaging an angiosarcoma in the periocular area. METHODS: We use laser speckle, hyperspectral, and photoacoustic imaging to monitor blood perfusion and oxygen saturation, as well as the molecular composition of the tissue. The information obtained from each imaging modality was combined in order to yield a more comprehensive picture of the function, as well as molecular composition of a rapidly growing cutaneous angiosarcoma in the periocular area. RESULTS: We found an increase in perfusion coupled with a reduction in oxygen saturation in the angiosarcoma. We could also extract the molecular composition of the angiosarcoma at a depth, depicting both the oxygen saturation and highlighting the presence of connective tissue via collagen. CONCLUSIONS: We demonstrate the different physiological parameters that can be obtained with the different techniques and how these can be combined to provide detailed 3D maps of the functional and molecular properties of tumors useful in preoperative assessment.

Is a bacterial biofilm in the lacrimal sac the cause of chronic refractory dacryocystitis? - A pilot study.

PURPOSE: A pilot study to identify bacterial biofilm in the lacrimal sacs of patients with chronic dacryocystitis, and in patients with epiphora but without discharge, using scanning electron microscopy. METHODS: Five patients: two with nasolacrimal duct obstruction without dacryocystitis, and three with dacryocystitis refractory to antibiotics, underwent external dacryocystorhinostomy. One control patient without infection was included. Bacterial cultures were obtained from the lumen of the lacrimal sac to analyze possible bacterial growth, including antibiotic resistance. Biopsies were taken from all lacrimal sacs and prepared for light and scanning electron microscopy. RESULTS: Scanning electron microscopy of all the lacrimal sac samples revealed structures consistent with bacterial communities and adjacent extracellular material, indicating biofilm formation. This was most prominent in one of the patients with chronic dacryocystitis. Bacteria were found not only on the luminal surface of the sac, but also within the tissue of the sac. Bacterial growth was identified in samples from two patients with chronic dacryocystitis, whereas samples from the other three patients showed no bacterial growth. CONCLUSION: Lack of patency of the lacrimal duct predisposes to bacterial growth, even in patients with no clinically confirmed infection of the lacrimal sac. The finding of a biofilm in patients with chronic dacryocystitis explains the lack of efficiency of antibiotic treatment at the concentrations used in clinical practice.

Outcomes of external and endonasal dacryocystorhinostomy according to a modified Lacrimal Symptom Questionnaire (Lac-Q).

Background: Nasolacrimal duct obstruction is usually treated using endoscopic or external dacryocystorhinostomy (DCR). The anatomic outcomes of both the endoscopic and external approaches are considered excellent. However, anatomic success does not translate into patient satisfaction. The current study assessed pre- and postoperative lacrimal problems using the symptom-based Lacrimal Symptom Questionnaire (Lac-Q) and investigated patient satisfaction depending on the choice of surgical technique. Methods: A total of 112 eligible patients with lacrimal problems treated using external or endonasal DCR at the ophthalmology and ear, nose, and throat clinics at Skane University Hospital, Scania, Sweden, over a four-year period, were enrolled in this retrospective study. Patients were considered eligible if they experienced preoperative epiphora and had lacrimal duct stenosis. They were offered treatment using either external or endonasal DCR and were allowed to freely choose the technique. Exclusion criteria consisted of previous ipsilateral DCR, congenital NLDO, age < 18 years, presence of cancer, previous orbital trauma, or noncompliance with postoperative follow-up. After surgery, the patients were sent the Lac-Q to evaluate their lacrimal symptoms pre- and postoperatively. Complementary questions were added pertaining to the operative scar and the patients' overall satisfaction with the operation. Results: In total, 67 (60%) patients with ages ranging from 18 to 88 years completed the questionnaire, 33 (49%) of whom underwent external DCR and 34 (51%) endonasal DCR. Of the 67 respondents, 51 (76%) were women and 16 (24%) were men. Patients scored preoperative lacrimal problems highly on the Lac-Q, reporting both symptomatic and social problems due to epiphora. Following surgery, the group that underwent external DCR remained home from work for 2 - 14 days (median, 3.5 days). However, 17 (52%) were retired. After the endonasal DCR, the patients remained home for 0 - 7 days (median, 2 days). Most patients were satisfied after DCR surgery, with both techniques significantly improving total, lacrimal symptom, and social impact scores (all P < 0.001). No differences in postoperative satisfaction were observed between the external DCR and endonasal DCR groups (P > 0.05). A small number of patients expressed scar-related concerns after external DCR. Conclusions: The patients perceived lacrimal problems as a significant symptomatic and social burden. Postoperative satisfaction and symptom relief were good regardless of the surgical approach. Further prospective studies assessing patient satisfaction and its correlation with anatomical and functional success rates after external and endonasal DCR could provide robust, practical, real-world implications.

First-Onset Dacryocystitis: Characterization, Treatment, and Prognosis.

INTRODUCTION: To assess the risk of recurrent dacryocystitis after first-onset dacryocystitis and to obtain a demographic profile and treatment characteristic for patients with first-onset dacryocystitis. METHODS: A retrospective study was performed on patients who had first-onset dacryocystitis during the years 2010-2013. Patients were followed up for 3 years. The analysis focused on the recurrence of dacryocystitis, demographics, medical treatment, and choice of lacrimal surgery. RESULTS: The inclusion criteria were met by 52 patients. Of these 15 (29%) had one or more recurrence of dacryocystitis, and 18 patients (34.6%) underwent lacrimal surgery. The mean age was 51.6 years (median 55.5, range 0-93). The female-to-male ratio was slightly under 3:1 (73.1%). The most frequent medical treatment was flucloxacillin capsules combined with chloramphenicol eye drops or ointment. CONCLUSIONS: The majority of patients with first-onset dacryocystitis had no further episodes of dacryocystitis. Some patients experienced recurrent and complicated infections requiring surgery and were thus a significant burden on the healthcare services. Various surgical options were used to clear the nasolacrimal obstruction causing dacryocystitis. Dacryocystorhinostomy was the most common procedure and showed excellent success rate.

Photoacoustic imaging of periorbital skin cancer ex vivo: unique spectral signatures of malignant melanoma, basal, and squamous cell carcinoma.

Radical excision of periorbital skin tumors is difficult without sacrificing excessive healthy tissue. Photoacoustic (PA) imaging is an emerging non-invasive biomedical imagi--ng modality that has potential for intraoperative micrographic control of surgical margins. This is the first study to assess the feasibility of PA imaging for the detection of periocular skin cancer. Eleven patients underwent surgical excision of periocular skin cancer, one of which was a malignant melanoma (MM), eight were basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs). Six tumors were located in the eyelid, and five in periocular skin. The excised samples, as well as healthy eyelid samples, were scanned with PA imaging postoperatively, using 59 wavelengths in the range 680-970 nm, to generate 3D multispectral images. Spectral unmixing was performed using endmember spectra for oxygenated and deoxygenated Hb, melanin, and collagen, to iden--tify the chromophore composition of tumors and healthy eyelid tissue. After PA scanning, the tumor samples were examined histopathologically using standard hematoxylin and eosin staining. The PA spectra of healthy eyelid tissue were dominated by melanin in the skin, oxygenated and deoxygenated hemoglobin in the orbicularis oculi muscle, and collagen in the tarsal plate. Multiwavelength 3D scanning provided spectral information on the three tumor types. The spectrum from the MM was primarily reconstructed by the endmember melanin, while the SCCs showed contributions primarily from melanin, but also HbR and collagen. BCCs showed contributions from all four endmembers with a predominance of HbO2 and HbR. PA imaging may be used to distinguish different kinds of periocular skin tumors, paving the way for future intraoperative micrographic control.

Laser Speckle Contrast Imaging of the Blood Perfusion in Glabellar Flaps Used to Repair Medial Canthal Defects.

BACKGROUND: The glabellar flap is a common technique for surgical repair after tumor excision in the medial canthal area. However, the outcome may be affected by partial flap necrosis. Little is known about the impact of surgery on blood perfusion and the postoperative course of reperfusion due to the absence of reliable and noninvasive perfusion monitoring techniques. The aim of this study was to use a modern imaging technique to assess blood perfusion in glabellar flaps. METHODS: Glabellar flaps were used to repair medial canthal defects following tumor excision in 7 patients. Blood perfusion was monitored using laser speckle contrast imaging: during surgery, immediately postoperatively (0 weeks), and at follow-up, 1, 3, and 6 weeks after surgery. RESULTS: Perfusion decreased gradually along the length of the flap, and reached a minimum 15 mm from the flap base. Perfusion in the proximal 20 mm of the flap was completely restored after 1 week, while the distal part of the flap was gradually reperfused over 6 weeks. Both the functional and esthetic surgical outcomes were excellent. CONCLUSIONS: The rapid reperfusion of the glabellar flap may be explained by its connection to the vascular network via the flap pedicle. In flaps longer than 20 mm, the distal part can be considered a free skin transplant, and a combination of a glabellar flap and a free skin graft could then be considered.

[Acute orbital trauma - risk of permanent impaired vision and double vision]. / Viktigt att agera snabbt vid traumatiska orbitala tillstånd.

The purpose of this article is to highlight the importance of appropriate management of acute orbital trauma, in order to prevent permanent impaired vision and double vision. Orbital blowout fractures with entrapment, so-called trapdoor fractures, occur more often in children than in adults, and require immediate surgical intervention. A blowout fracture with impingement, so-called open door fracture, does not require immediate intervention. However, if the motility does not improve within 1-2 weeks surgery may be required. The symptomatology in children with orbital fractures may differ significantly from that in adults, and vasovagal symptoms such as bradycardia and nausea are common.  Orbital compartment syndrome is characterized by an acute increase in orbital pressure, and may result in irreversible blindness if not promptly treated with lateral canthotomy and cantholysis.

Laser Speckle Contrast Imaging of a Rotational Full-Thickness Lower Eyelid Flap Shows Satisfactory Blood Perfusion.

Full-thickness eyelid flaps from the lower eyelid are frequently used to repair larger upper eyelid defects. Perfusion monitoring has recently been implemented in several reconstructive surgical procedures, however, perfusion monitoring of a rotational eyelid flap has not yet been described. The authors' employed laser speckle contrast imaging to monitor blood perfusion in a rotational flap from the lower eyelid, used to cover a large tumor defect in the upper eyelid. Perfusion in the flap decreased by only 50% during surgery and was almost completely restored 5 weeks later at flap division (91%). The excellent surgical outcome in the present case is deemed to be the result of satisfactory blood perfusion of the flap.

Reperfusion of Free Full-Thickness Skin Grafts in Periocular Reconstructive Surgery Monitored Using Laser Speckle Contrast Imaging.

PURPOSE: Free skin grafts are frequently used in reconstructive surgery. However, little is known about the course of reperfusion due to the previous lack of reliable perfusion monitoring techniques. The aim of this study was to use state-of-the-art laser speckle contrast imaging to monitor free skin grafts in the periocular area. METHODS: Seven patients needing surgery due to tumor removal or cicatricial ectropion in the periocular region underwent reconstructive surgery using free skin grafts from either the contralateral upper eyelid or the upper inner arm. The free skin grafts measured 10-30 mm horizontally and 9-30 mm vertically. Blood perfusion was monitored using laser speckle contrast imaging immediately postoperatively (0 weeks) and at follow-up after 1, 3, and 7 weeks. RESULTS: All grafts were reperfused gradually during healing, the median value being 46% in the central part of the graft after 1 week and 79% after 3 weeks. The grafts were completely reperfused after 7 weeks. No difference was observed in the rate of reperfusion between the center and periphery of the grafts (p = not significant). The cosmetic and functional outcome was excellent in all but 1 patient, who developed ectropion that had to be surgically corrected. CONCLUSIONS: Skin grafts in the periorbital area are fully reperfused after 7 weeks. The periocular area is known to be well-vascularized and thus forgiving to reconstructive surgery. Future investigations of the reperfusion of free skin grafts in other parts of the body or in higher-risk populations should be carried out.

Novel Evidence Concerning Lacrimal Sac Movement Using Ultra-High-Frequency Ultrasound Examinations of Lacrimal Drainage Systems.

PURPOSE: Current hypothesis regarding the mechanism of active tear drainage is based on studies performed ex vivo or under nonphysiological conditions. Novel ultra-high-frequency ultrasound has the advantage of generating images with superior resolution, enabling measurements of low flow in small vessels, and the tracking of tissue motion in real time. The purpose of this study was to investigate the lacrimal drainage system and active drainage using this modality. METHODS: The upper lacrimal drainage system was investigated with 40-70 MHz ultrasound in 22 eyes in 13 patients. Irrigation confirmed a lacrimal obstruction in 10 eyes. Motion tracking was used to map movement of the lateral lacrimal sac wall and to measure flow when possible. RESULTS: The anatomy of the upper lacrimal drainage system was mapped in vivo, including the proximal canaliculi, which have not previously been imaged. The lacrimal sac lumen is slit shaped in its resting state but is distended when irrigated or if a nasolacrimal duct obstruction is present. Thus, the healthy lacrimal sac is not a cavity, and the medial retinaculum does not act against a stretched structure. Motion tracking visualized the "lacrimal pump," showing that the direction of motion of the lateral lacrimal sac wall is mainly in the sagittal plane during blinking. CONCLUSIONS: Ultra-high-frequency ultrasound allows detailed physiological monitoring of the upper lacrimal drainage system in vivo. Our findings suggest that current theories of active tear drainage need to be reappraised.

Successful Free Bilamellar Eyelid Grafts for the Repair of Upper and Lower Eyelid Defects in Patients and Laser Speckle Contrast Imaging of Revascularization.

PURPOSE: It is generally believed that large eyelid defects must be repaired using a vascularized flap for 1 lamella, while the other can be a free graft. Recent studies indicate that the pedicle of a tarsoconjunctival flap does not contribute to blood perfusion. The purpose of this study was to explore whether large eyelid defects can be repaired using a free bilamellar eyelid autograft alone. METHODS: Ten large upper and lower eyelid defects resulting from tumor excision were reconstructed using bilamellar grafts harvested from the contralateral or opposing eyelid. Revascularization of the flap was monitored during healing using laser speckle contrast imaging, and the surgical outcome was assessed. RESULTS: The functional and cosmetic results were excellent. All grafts survived and there was no tissue necrosis. Only 1 patient underwent revision after 4 days as the sutures came loose. Two patients developed minimal ectropion but needed no reoperation. All patients were satisfied with the surgical results. Perfusion monitoring showed that the grafts were gradually revascularized, exhibiting 50% perfusion after 4 weeks and 90% perfusion after 8 weeks. CONCLUSIONS: A free bilamellar eyelid graft appears to be an excellent alternative to the tarsoconjunctival flap procedure in the reconstruction of both upper and lower eyelid defects, especially in patients who cannot tolerate visual axis occlusion or the 2-stage procedure of the conventional staged flap procedure.

Revascularization After H-plasty Reconstructive Surgery in the Periorbital Region Monitored With Laser Speckle Contrast Imaging.

BACKGROUND: H-plasty reconstructive surgery is commonly used to close defects after tumor excision in the periorbital region. Revascularization of the bipedicle skin flaps is essential for healing. However, it has not previously been possible to study this revascularization in humans due to the lack of noninvasive perfusion monitoring techniques. The aim was to monitor perfusion in H-plasty flaps during surgery and during postoperative follow-up, using laser speckle contrast imaging. METHOD: H-plasty, i.e., bipedicle random advancement skin flaps, was used for reconstruction of the eyelids after tumor removal in 7 patients. The median length and width of the skin flaps were 13 mm (range, 8-20 mm) and 10 mm (range, 5-11 mm), respectively. Blood perfusion was measured using laser speckle contrast imaging during surgery and at follow up 1, 3, and 6 weeks postoperatively, to monitor revascularization. RESULTS: Immediately postoperatively, the perfusion in the distal end of the flaps had fallen to 54% (95% CI, 38%-67%). The perfusion then quickly increased during the healing process, being 104% (86%-124%) after 1 week, 115% (94%-129%) after 3 weeks, and 112% (96%-137%) after 6 weeks. There was no clinically observable ischemia or tissue necrosis. CONCLUSIONS: Revascularization of the H-plasty procedure flaps occurs quickly, within a week postoperatively, presumably due to the existing vascular network of the flap pedicle, and was not dependent on significant angiogenesis. This perfusion study confirms the general opinion that H-plasty is a good reconstructive technique, especially in the periorbital region with its rich vascular supply.

Revascularization of Free Skin Grafts Overlying Modified Hughes Tarsoconjunctival Flaps Monitored Using Laser-Based Techniques.

PURPOSE: It has recently been shown that the flap pedicle does not supply blood to a tarsoconjunctival graft in the modified Hughes procedure in patients. This raises questions concerning the rate of revascularization of the free skin graft commonly used to reconstruct the anterior lamella. The aim of this study was, thus, to monitor the course of revascularization in free skin grafts overlying modified Hughes tarsoconjunctival flaps, using laser-based techniques. METHODS: Free skin grafts from the upper eyelid or upper arm in 9 patients were used to cover a tarsoconjunctival flap according to the modified Hughes procedure. Blood perfusion was monitored using laser speckle contrast imaging, and vascular reactivity was studied with laser Doppler velocimetry after heating the tissue to 44°C. Measurements were made at the time of surgery (baseline) and at 1, 3, 8, and 16 weeks postoperatively. RESULTS: The gradual increase in perfusion of the free skin grafts during the healing process indicates revascularization. A slight increase in perfusion was seen already after 1 week. Perfusion reached 50% of the baseline after 3 weeks, and complete restoration of perfusion was seen after 8 weeks. The vascular function monitored with heat-induced hyperemia increased in a similar fashion. CONCLUSIONS: Full-thickness skin grafts revascularize within 3 to 8 weeks, despite overlying a tarsoconjunctival flap, which has recently been reported to be avascular. This provides further evidence that it should be possible to repair large eyelid defects using free full-thickness eyelid grafts.

Perfusion Monitoring Shows Minimal Blood Flow From the Flap Pedicle to the Tarsoconjunctival Flap.

BACKGROUND: A previous study in pigs has shown that the pedicle of the tarsoconjunctival flap does not appear to have adequate blood perfusion. The aim of this study was to monitor perfusion in tarsoconjunctival flaps in patients with large lower eyelid defects resulting from tumor surgery. METHODS: The modified Hughes procedure was performed in 13 patients. Blood perfusion was monitored using laser Doppler velocimetry and laser speckle-contrast imaging. RESULTS: Blood flow decreased gradually from the pedicle base to the end of the flap and was 19% at the flap base, 11% in the middle of the flap, and 4% in the distal end of the flap. The flaps survived, and there was no tissue necrosis. CONCLUSIONS: Tarsoconjunctival tissue survival does not seem to be dependent on a conjunctival flap. Free tarsoconjunctival grafts or composite grafts might be considered as viable alternatives in reconstruction of major eyelid defects.

Large Eyelid Defect Repair Using a Free Full-Thickness Eyelid Graft.

Large eyelid full-thickness defects are traditionally repaired using flaps with a blood-supplying pedicle, for the reconstruction of the anterior or posterior lamella or both. This is a 2-stage procedure involving occlusion of vision in the affected eye for 4-8 weeks, as the flap pedicle is not divided until vascularization is deemed adequate. However, the importance of using a flap with a pedicle to ensure adequate perfusion of the graft has recently been questioned.

Clinical, optical coherence tomography, and fundus autofluorescence findings in patients with intraocular tumors.

PURPOSE: To describe clinical, optical coherence tomography (OCT) and fundus autofluorescence (FAF) findings in patients with intraocular tumors and determine if OCT and FAF could be helpful in the differential diagnosis and management of different choroidal tumors. METHODS: Forty-nine patients with untreated, macular, midperipheral, and extrapapillary intraocular tumors were included. All patients underwent ophthalmic examination: best-corrected visual acuity, slit-lamp biomicroscopy, funduscopy, and standardized B mode, and if possible A mode, ultrasonography, and OCT and FAF imaging of the surface of the intraocular tumors. RESULTS: Of the 49 patients studied, 19 had choroidal nevi, ten had indeterminate choroidal melanocytic lesions (IMLs), ten had malignant melanomas, and ten had other choroidal tumors. The choroidal nevi revealed subretinal fluid (SRF) on OCT in only 11%. FAF detected isoauto-fluorescence in 42%, hypoautofluorescence in 37%, patchy FAF pattern in 16%, and a diffuse FAF pattern in 5%. Seventy percent of patients with IML showed SRF on OCT and 20% showed tumor growth on follow-up, detected only by OCT and FAF imaging. FAF revealed a patchy pattern in 50% and a diffuse pattern in 40% of cases with IML. Ninety percent of the patients with choroidal melanoma had SRF on OCT and FAF revealed a patchy pattern in 60% and a diffuse pattern in 40%. Patients with other choroidal tumors had SRF on OCT in 30% of cases and no characteristic pattern on FAF. CONCLUSION: Both OCT and FAF were helpful in the differential diagnosis of choroidal nevi versus IMLs, choroidal melanomas, and other choroidal tumors. Also, detailed and periodical clinical evaluation of patients with intraocular tumors using OCT and FAF imaging for the detection of both SRF and FAF patterns overlying the tumor can be useful for detection of tumor growth.

Scaffolding the retina: the interstitial extracellular matrix during rat retinal development.

PURPOSE: To examine the expression of interstitial extracellular matrix components and their role during retinal development. MATERIAL AND METHODS: Fibronectin (FN), collagen IV (Coll IV) and laminin 5 (Lam 5) expression in rat retinas from developmental stages E17 to adult were studied. In addition, PN5 full-thickness retinas were cultured for 7 days with dispase, which selectively cleaves FN and Coll IV, at either 0.5 U/ml or 5.0 U/ml for 3 or 24h. Eyecups and retinal cultures were examined morphologically using hematoxylin and eosin staining and immunohistochemistry. RESULTS: Coll IV, Lam 5 and FN were all transiently expressed in the interstitial matrix of the retinal layers during development. The retinal layers in dispase treated explants was severely disturbed in a dose and time dependent manner. CONCLUSIONS: FN, Lam 5 and Coll IV, are present in the interstitial extracellular matrix during rat retinal development. Enzymatic cleavage of FN and Coll IV early in the lamination process disrupts the retinal layers implicating their pivotal role in this process.

Ocular phenotype analysis of a family with biallelic mutations in the BEST1 gene.

PURPOSE: To investigate the genetic cause and perform a comprehensive clinical analysis of a Danish family with autosomal recessive bestrophinopathy; to investigate whether Bestrophin may be expressed in normal human retina. DESIGN: Retrospective clinical and molecular genetic analysis and immunohistochemical observational study. METHODS: setting: National referral center. participants: A family with 5 individuals and biallelic BEST1 mutations, and enucleated eyes from 2 individuals with nonaffected retinas. observation procedures: Molecular genetic analysis included sequencing of BEST1 and co-segregation analysis. Clinical investigations included electro-oculography, full-field electroretinography, multifocal electroretinography, spectral-domain optical coherence tomography, and fundus autofluorescence imaging. Immunohistochemical analysis was performed. main outcome measures: BEST1 mutations, imaging findings, electroretinography amplitudes, and implicit times. RESULTS: The index case was compound heterozygous for p.A195V and a novel 15 base pair deletion leading to p.Q238L. The index case at age 10 demonstrated multifocal vitelliform changes that were hyperautofluorescent, cystoid macular edema in the inner nuclear layer, no light rise in the electro-oculography, and a reduced central but preserved peripheral retinal function by multifocal electroretinography. Full-field electroretinography demonstrated a reduced rod response and inner retina dysfunction. Retinal structure was normal in all 3 family members who carried a sequence change in BEST1. Electro-oculography light peak was reduced in both the mother and sister (heterozygous for p.Q238L). Immunohistochemistry could not confirm the presence of Bestrophin in normal human retina. CONCLUSIONS: Because of a relatively well preserved retinal function, autosomal recessive bestrophinopathy may be a suitable first candidate, among the BEST1-related ocular conditions, for gene replacement therapy.

Effects of glial cell line-derived neurotrophic factor on the cultured adult full-thickness porcine retina.

BACKGROUND: The tissue culture system offers a possibility to study factors involved in neuronal survival which may be important in a transplantation paradigm. The use of adult tissue in this setting poses specific challenges since traditionally mature neurons survive poorly in vitro. In the current paper, we have explored effects of glial cell line-derived neurotrophic factor (GDNF) on cultures of adult porcine retina. METHODS: Full-thickness retinal sheets were isolated from adult porcine eyes and were cultured for 5 or 10 days under standard culture conditions with or without GDNF added to the culture medium. The grafts were analyzed morphologically using hematoxylin and eosin staining, immunohistochemistry and transferase dUTP nick end labeling (TUNEL) labeling. Retinas derived from normal adult porcine eyes were used as controls. RESULTS: After 5 d in vitro (DIV), cultures without GDNF showed dissolving retinal lamination while specimens cultured with GDNF displayed the normal laminated morphology. At 10 DIV, the untreated cultures had been reduced to a degenerated cell mass, while the GDNF-cultured specimens retained thin but distinguishable retinal layers. TUNEL labeling confirmed these results. Immunohistochemical labelings and outer nuclear layer thickness measurements showed an increased preservation of photoreceptors and horizontal cells in the GDNF-treated group. CONCLUSIONS: The procedure of culturing retina involves several steps causing severe traumatic effects on the tissue, such as ganglion cell axotomy, interruption of the blood flow as well as separation from the retinal pigment epithelium (RPE). In this paper, we have shown that addition of GDNF in the culture medium attenuates the effect of these steps, resulting in enhanced preservation of several retinal neuronal subtypes. The results may be of importance for research in retinal transplantation where storage time of the donor tissue prior to transplantation is a critical issue.