Free-hand Dissection Research Articles

Novel technique of tetra trephination for elliptical-shaped tectonic patch grafts in peripheral sterile keratolysis.

To describe a novel technique of tectonic patch grafts for elliptical-shaped peripheral sterile keratolysis with iris prolapse. We performed a full thickness corneo-scleral patch graft in five eyes of five patients with elliptical-shaped peripheral sterile keratolysis with perforation and iris tissue prolapse. In this technique, four disposable trephines with predetermined diameter were employed to fashion both the host and the donor without any free-hand dissection. An intact anatomical integrity of the globe without the need for any repeat tectonic measures was considered as a successful outcome. The mean age was 34.2 ± 10.2 years (18-45). The mean total surgical time and donor preparation time was 94.4 ± 7.12 min and 7.6 ± 1.14 min, respectively. The intraoperative course was uneventful in all cases and all eyes had a well-maintained corneal integrity till 6 months follow up. We describe a simple, reproducible, and time-saving technique of performing elliptical shaped corneoscleral patch grafts for peripheral corneal perforations with iris prolapse.

Tectonic lamellar keratoplasty: simplified management of corneal perforations with an automated microkeratome

ObjectiveTo describe a simplified tectonic lamellar keratoplasty (LK) technique, which can be used to manage full-thickness corneal perforations. DesignCase series demonstrating a new surgical technique. ParticipantsA 69-year-old female with rosacea keratitis and peripheral corneal perforation; a 49-year-old male with keratoconus, ipsilateral herpes simplex keratitis and an inferonasal corneal perforation; and a 12-year-old female with an apparently spontaneous central corneal perforation. ResultsThese 3 patients with full-thickness corneal perforations were successfully managed with this automated tectonic LK technique. ConclusionsTectonic LK has benefits relative to tectonic penetrating keratoplasty but remains unpopular because it is technically difficult to prepare the host and donor corneas. This tectonic LK technique negates the need for challenging free-hand dissection and gives an excellent donor fit to the host bed. This provides an easier and more effective way to manage corneal perforations.

Acoustic communication and burrow acoustics are reflected in the ear morphology of the coruro (Spalacopus cyanus, Octodontidae), a social fossorial rodent

We studied the middle and inner ears of seven adult coruros (Spalacopus cyanus), subterranean and social rodents from central Chile, using free-hand dissection and routine staining techniques. Middle ear parameters that were focused on here (enlarged bullae and eardrums, ossicles of the "freely mobile type") are believed to enhance hearing sensitivity at lower frequencies. The organ of Corti was of a common mammalian type and revealed three peaks of higher inner hair cell densities. Based on a position frequency map, frequencies were assigned to the respective peaks along the basilar membrane. The first peak at around 300-400 Hz is discussed with respect to the burrow acoustics, while the peak around 10-20 kHz is probably a plesiomorphic feature. The most pronounced peak at around 2 kHz reflects the frequency at which the main energy of vocal communication occurs. The morphology of the ear of the coruro corresponds to the typical pattern seen in subterranean rodents (low frequency and low-sensitivity hearers), yet, at the same time, it also deviates from it in several functionally relevant features.

Self-Illuminated Contact Lens for Peripheral Vitreous Surgery

A modified self-retaining plano-concave contact lens for visualization of the ora serrata and anterior vitreous during vitreous surgery is described. A groove to hold a light pipe is made in the standard self-retaining, plano-concave, one-piece acrylic lens with footplates. A drop of viscoelastic material between the lens and the cornea holds the lens in place by suction. The groove (1 mm in depth and diameter) supports the tip of the endoillumination probe held by an assistant. This illuminates the area while the surgeon indents the sclera with the free hand, facilitating quick and well-controlled vitreous base dissection. This system offers a simple, effective, and quick method for illumination of the ora serrata and anterior vitreous in aphakic and pseudophakic eyes during vitrectomy.

Immunocytochemical Study of α 1 and β 2/3 Subunits of GABA A Receptors in Freehand Isolated Vestibular Deiters' Neurons

Vestibular Deiters' neurons have been isolated from bovine brain by the Hydén's freehand dissection technique and challenged with monoclonal antibodies directed toward the alpha 1 and beta 2/3 subunits of the GABAA receptors. Subsequent challenge with fluorescent secondary antibodies and confocal microscopy allowed the study of the cellular distribution of such subunits. In Deiters' neurons the beta 2/3 subunit displayed a clear presence all along the cell body profile and the initial parts of the dendrites. The alpha 1 subunit was found highly present all over the cell interior except the nuclear profiles. The strong presence inside the cells possibly masked its presence on the plasma membrane. However, in part of the cells studied a distinct presence on the plasma membrane was evident. This subunit was visualized also all along the long dendrites of these neurons. The approach we describe here, involving freehand isolated mature neurons from adult animals, may allow a better characterization of the tridimensional distribution of different types of neuronal GABAA receptors in the respect of the approach with brain slices.

Microkeratome-based limbal harvester for limbal stem cell transplantation: preliminary studies

PURPOSE: To develop a mechanical device to perform limbal transplantation. This procedure is a valuable surgical technique for management of limbal stem cell deficiency. However, the freehand dissection to obtain donor tissue is laborious and time-consuming. METHODS: A manual microkeratome (LSK One; Moria/Microtech, Doylestown, Pennsylvania) using a redesigned head (200 μm thickness, 16-mm blade) was used to create partial thickness corneoscleral caps from human donor globes. RESULTS: Corneoscleral caps obtained from human donor globes included a ring of approximately 1 mm of perilimbal sclera in contiguity to the cornea. Scanning electron microscopy showed a smooth cut surface with some chatter at the margins. CONCLUSION: This instrument ensures an effective and straightforward method to dissect the area where stem cells have been indirectly localized.

Quantification and localization of hyaluronan in a PTFE polymer implanted in the corneal stroma.

The amount and distribution of hyaluronan in a PTFE polymer used to support an artificial cornea implanted in the rabbit cornea were determined. The findings were used to describe the polymer-corneal stroma interface and the reason for the translucence and wettability of this originally opaque and hydrophobic biomaterial. PTFE disks (6 mm in diameter, 0.2 mm thick, 50 microns in pore size) were implanted after a free-hand intralamellar dissection. The corneas were removed 15 days, 1 month, and 3 months after implantation. The hyaluronan content of pepsin-solubilized corneal stromal extracts and its distribution (7 microns cryostat sections) were investigated using an alkaline phosphatase-linked hyaluronectin assay that specifically detects nanogram amounts of hyaluronan. A PTFE polymer implant caused large, transient increases in hyaluronan density in the implanted stroma. The presence of amphiphilic hyaluronan in the polymer 15 days post implantation probably produced translucence and wettability of this opaque, hydrophobic implant despite the absence of cells. The hyaluronan density in the PTFE polymer increased considerably during the first month and then decreased to stabilize at a moderate level by the third month. These changes in hyaluronan density parallel the invasion of the polymer by inflammatory cells during the first month and the subsequent replacement of these cells by fibroblasts. The PTFE polymer is a good interface that is compatible with the native corneal stroma, and our results indicate that hyaluronan, because of its amphiphilic character, plays a major role in the polymer wettability and translucence and in the production of typical corneal extracellular matrix within the pores of the polymer.

Relaxing Incisions With Compression Sutures to Reduce Astigmatism After Epikeratoplasty

Relaxing incisions with compression sutures were performed in seven eyes with high astigmatism following epikeratoplasty for keratoconus and in one case of posttraumatic aphakia. Mean preoperative keratometric astigmatism was 7.64 +/- 2.51 diopters (range 5.50 D to 13.00 D) in the epikeratoplasties for keratoconus and about 10.00 D in the hyperopic epikeratoplasty. The surgical procedure consisted of a free-hand dissection perpendicular to the steeper meridian along the scar between the edge of the epikeratoplasty lenticule and the recipient cornea, with an additional incision into the recipient stroma to an approximate depth of 80%. Following the incisions, compression sutures were added 90 degrees away in the flatter meridian. After surgery, the net decrease in keratometric astigmatism was 6.50 D +/- 2.90 D (range 5.00 to 13.00 D) in the eyes with epikeratoplasty for keratoconus and 6.50 D in the eye with hyperopic epikeratoplasty. Uncorrected visual acuity improved in six eyes and remained unchanged in two eyes. Spectacle-corrected visual acuity improved in every eye and contact-lens-corrected visual acuity improved in seven eyes and was unchanged in one eye. This procedure, already employed for astigmatism correction after penetrating keratoplasty, was effective in decreasing astigmatism after epikeratoplasty.

Peculiarities of the Polypeptide Composition of the Morphofunctionally Different Neurons of the Rat Brain

The present study was aimed to identify peculiarities of the polypeptide composition in three morphofunctionally different classes of neurons of the rat brainstem: giant multipolar neurons of n. reticularis gigantocellularis, relay sensory neurons of lateral geniculate body and pyramidal neurons of the pyramidal layer of the CA3 - CA4 fields of hippocampus. The method of free hand dissection of neurons and sodium dodecyl sulfate (SDS) slab gel microelectrophoresis in our modifications were used. Polypeptide peculiarities for each class of investigated neurons have been found. Most of these polypeptides belong to the low molecular weight range (8.8-24.3 KD) of the polypeptide spectra and only some of them to the high one (64.6-75.3 KD).

Preliminary Evaluation of the Use of Mussel Adhesive Protein in Experimental Epikeratoplasty

We have used a preliminary formulation of a bioadhesive in an experimental model of epikeratoplasty in rabbits. The adhesive, termed mussel adhesive protein (MAP), is a repeating decapeptide polymer that is the natural adhesive substance produced by the common blue mussel (Mytilus edulis) and is used here in conjunction with an enzymatic cross-linking agent. This study marked the first in vivo use of this material in adhering ophthalmic tissue planes. We used a simplified epikeratoplasty technique highlighted by freehand dissection of donor lenticules and host keratotomies. Approximately 10 microL of a combination of MAP and the cross-linking agent was applied directly to the host cornea and the donor lenticules were secured into the keratotomies using eight interrupted 10-0 nylon sutures. All of the sutures were removed 72 hours postoperatively. Eleven of the 15 animals retained their epikeratoplasty lenticules throughout the entire postoperative period. Four animals had initially intact lenticules that sloughed within the first postoperative week; this sloughing, we believe, was attributable to difficulties in tucking irregularly thickened lenticule edges into the keratotomy. In control animals that had lenticules secured with sutures alone, suture removal at 72 hours consistently produced immediate lenticule sloughing. Clinical examinations and histopathologic studies disclosed no untoward effects of the adhesive on the donor or host corneal tissue. We believe that this preliminary study indicates a potential adjunctive role for MAP in epikeratoplasty.

In vitro effect of danazol on steroidogenesis in developing human follicles. An interim report.

In order to show whether or not danazol exerts direct effects on the cells of the human ovarian follicle, follicular granulosa and thecal cells of follicles in different stages of development are currently being investigated in short-term incubation experiments and during more prolonged cell culture. Cells are isolated by free-hand dissection from follicles of 8 to 25 mm diameter, corresponding approximately to cycle day 7 to 14. Granulosa and thecal cells have so far been incubated for 2 to 4 hours in the presence or absence of hCG and/or danazol. A small number of cell cultures of granulosa cells have been performed for up to 6 days in the presence or absence of danazol, hFSH, and hLH, in various combinations. The progesterone (P), estradiol-17 beta (E) and androstenedione (A) content of the incubation or culture medium and the tissue content of cyclic AMP (cAMP) were measured. Danazol (10 micrograms/ml) inhibited thecal cAMP production in most short-term experiments but only the hCG-stimulated cAMP production of granulosa cells. Short-term production of P and E by granulosa and of A and E by thecal cells was also inhibited by danazol in certain circumstances. In tissue cultures of granulosa cells lasting up to 4 days, danazol (1 and 10 micrograms/ml) inhibited both the FSH and LH-stimulated P production. Provided that any unspecific effects of the concentrations of danazol used can be ruled out, the results seem to indicate that this substance may act directly upon steroidogenesis at the ovarian follicular level.

Acute effects of gonadotrophins and prostaglandins on the metabolism of isolated ovarian follicles from PMSG-treated immature rats.

ABSTRACT Pre-ovulatory follicles from immature rats injected with a low dose of PMSG have been used for studies of carbohydrate and protein metabolism in vitro. The follicles were isolated by free-hand dissection and incubated in modified Krebs bicarbonate buffer for periods up to 4 h. The lactic acid content of the medium was analyzed after various incubation periods. In most experiments the tissue uptake of [14C]α-amino-isobutyric acid and [3H] leucine as well as the incorporation of [3H] leucine into follicular protein were determined. Addition of LH or FSH to the incubation medium markedly stimulated the production of lactic acid, whereas prostaglandins of the E-type were less effective. Prostaglandin F2α and dibutyryl cyclic 3′, 5′-AMP had no effect at all. No in vitro effects were seen on the uptake and incorporation af labelled amino acids. When LH or FSH was injected to the animals in vivo 1 or 2 h before incubation of the follicles, the lactic acid production was also stimulated, but only the injection of LH stimulated the protein incorporation of [3H] leucine.

Effects of gonadotrophins, dibutyryl cyclic AMP and prostaglandin E2 on amino acid transport and glycolysis in isolated rat corpora lutea.

AbstractCorpora lutea (CL) were isolated by free hand dissection from ovaries of 34–39 day‐old Sprague‐Dawley rats injected with 10 IU Pregnant Mare's Serum gonadotropin (PMSG) when 30 days old leacling to ovulation in the morning of day 33. Injection of luteinizing hormone (LH) to rats 2 h before removal of the ovaries stimulated the in vitro uptake of the non‐utilizable amino acids, a‐aminoisobutyric acid (AIB‐14C) and 1‐amino‐cyclopentane‐carboxylic acid (cycloleucine‐3H) by 3‐day‐old CL during a subsequent incubation in Krebs bicarbonate buffer. Prolactin and FSH were without effects. Addition of LH directly to the incubation medium did not stimulate amino acid uptake of the isolated CL, while addition of dibutyryl cyclic AMP (dbcAMP) enhanced this uptake. Glycolysis was estimated by determination of lactic acid accumulation in die incubation medium. When LH was injected to the animals 2 h before sacrifice, lactic acid production of 3‐day‐old CL was stimulated during a subsequent incubation period. Addition of LH in vitro caused a slight increase in the rate of glycolysis, and dbcAMP and prostaglanclin E2 (PGE2) also exhibited stimulatory effects on lactic acid production by the incubated CL. In a few experiments rats with 7‐day‐oId CL were given injections of LH. While amino acid uptake was not stimulated in these old CL, LH did enhance the rate of glycolysis in these CL.

Enzymatic studies of glucose phosphorylation in the glucagon-producing cells of duck pancreas.

The glucagon-producing A2-cells of the pancreatic islets seem to show a reversed secretory response to glucose in comparison with the insulin-producing B-cells. In this investigation, the glucose phosphorylating process has been studied in A2-cells isolated from cryostat sections of duck pancreases. The cell samples were prepared by free-hand dissection in cold heptane during observation with a dark field stereo microscope.

Nuclear and cytoplasmic RNA in normal and dystrophic neurons

Base ratios of RNA were determined separately on nuclei and cytoplasm from anterior horn cells obtained by free-hand dissection. The nuclei were separated from the cytoplasm by micromanipulation and the extraction was done with ribonuclease followed by microhydrolysis, microelectrophoresis and microdensitometry.

Penetration of exogenous testosterone, pregnenolone, progesterone and cholesterol into the seminiferous tubules of the rat.

Tritiated cholesterol, pregnenolone, progesterone and testosterone were injected intravenously into adult male rats. They were killed from 30 min to 10 days after the injection of cholesterol, and from 2 to 30 min after the other steroids. The interstitial tissue of the testis was separated by free hand dissection from the seminiferous tubules. The tissue samples were dried, weighed, combusted in closed glass ampules and the tritium gas was subsequently counted in a metal cathode internal gas counter. Pregnenolone, progesterone and testosterone behaved equally, producing maximal radioactivity in both tissue compartments between 2 and 5 min after the injection, but the maximal radioactivity appeared at 24 hr after the cholesterol injection. The radioactivity in the seminiferous tubules was always significantly lower than in the interstitial tissue, the difference being most marked in the case of cholesterol. Very low or negligible counts were obtained in the seminiferous tubules throughout the cholesterol ...

Respiration of isolated parathyroid cells from nephrectomized and vitamin D-treated rats.

Parathyroid cells from adult female rats were isolated by free-hand dissection and oxygen uptake of these cells was determined using succinate as substrate. The micro-diver technique (6) proved suitable for studies concerned with respiratory function of isolated parathyroid cells under various experimental conditions. Parathyroid cells were obtained from control, nephrectomized and vitamin D-treated rats. The respiration of parathyroid cells from animals nephrectomized 4 and 24 hr earlier was slightly lower than in cells from unoperated controls, while 48 hr postoperatively there was a significant increase in respiratory rate of these cells. Histochemical determinations of succinic dehydrogenase in parathyroid glands showed greater reactivity 24 and 48 hr after nephrectomy. Vitamin D treatment for periods varying between 1 and 5 days was followed by a gradual decrease in oxygen uptake of the parathyroid cells and a slight decrease in histochemically determined succinic dehydrogenase activity. (Endocrinolo...

Submicroscopic structure of single neurons isolated from rabbit lateral vestibular nucleus

Giant neurons from the lateral vestibular nuclei of rabbits have been isolated in 0.25 M sucrose by freehand dissection. They were fixed by immersion in glutaraldehyde followed by osmium tetroxide and dehydrated with Durcupan A prior to embedding in Araldite 502. By means of this simple method it was possible to achieve a preservation of isolated cells which was comparable to that obtained when small pieces of tissue are preserved, sectioned, and examined in the electron microscope. Mitochondria, however, were characterized by a condensed, granular matrix and more closely resembled those found in fractions of cells homogenized in 0.25 M sucrose than in tissue sections. Plasma membranes were essentially intact, and in fortuitous sections a trilaminar structure was demonstrated. These data support the assumption that disruption of plasma membranes and other morphological changes previously reported occur primarily after isolation of nerve cells and during their preparation for examination in the electron microscope. Although these changes could be minimized, it was difficult to estimate accurately their extent.

Isolation and embedding of single nerve cells for electron microscopy.

Individual neurons have been isolated by freehand dissection under binocular, 40-100 power magnification from the lateral vestibular nucleus of fresh rabbit brain. The cells were fixed with glutaraldehyde and OsO4 and dehydrated in 33%, 50%, 60% (v/v) and pure Durcupan A prior to embedding in Araldite 502. Blocks were cast in lids of BEEM capsules. The locating of the individually embedded cells and trimming of the blocks were facilitated by means of a holder that permitted visualization in both diffuse transmitted and incident light.

Action of gonadotrophins in vitro on the respiration of isolated interstitial cells from the testis of the rat.

A technique for freehand dissection of interstitial cells from the testis of the rat is described. Isolated clusters of interstitial cells have been identified histochemically by the method of Wattenberg (15) and their masses determined by the direct X-ray absorption technique of Rosengren (18). The isolated interstitial cells have been used for in vitro experiments in the measurement of oxidation of succinate under the influence of gonadotrophins using Zeuthen's micro-diver technique (14). The respiratory rate of these cells was significantly raised when LH (NIH-LH-B3) was added to the medium in concentrations varying between 30 and 70 μg/ml. No stimulatory effect in oxygen uptake was found by FSH (NIH-FSH-S2) in vitro in a concentration of 50 μg/ml. The method described thus seems suitable for further studies concerning the influence of gonadotrophins on the various cell components of the testis. (Endocrinology 83: 855, 1968)