Microsurgical Method Research Articles

Selective excimer laser ablation of the trabecular meshwork. Clinical results

Examination of the efficacy of pore formation in the trabecular meshwork by excimer laser to reduce intraocular pressure in glaucoma eyes. In 27 consecutive eyes with chronic simple glaucoma and 8 eyes with low-tension glaucoma, 3 to 5 pores were ablated into the trabecular meshwork with an excimer laser (308 nm, 35-55 mJ/mm2), creating an open communication between the anterior chamber and Schlemm's canal. This was accomplished by the use of a 400-micron quartz fiber and a modified Trokel goniolens. All patients were candidates for trabeculectomy because visual fields continued to deteriorate in spite of maximum medication. Intraocular pressure was median reduced by 7 mmHg (range 10.5 to 1.5 mmHg) in 22 of 27 eyes with chronic simple glaucoma over a median follow-up of 7 months. In 12 eyes, further medication has to be continued, yet at a lower dose and lower level of intraocular pressure. In five eyes therapy failed. In three of these eyes, a trabeculectomy had to be performed. In eight eyes with low-tension glaucoma, a median reduction of intraocular pressure of 5 mmHg (range 10 to 0.5 mmHg) was accomplished over a median follow-up of 7 months. In five of these eyes, further medication on a lower level was continued. No further surgery was necessary. With the microsurgical method of pinpoint ablation of the trabecular meshwork by excimer laser, intraocular pressure was reduced in 30 of 35 eyes over a median follow-up period of 7 months. These results encourage us to continue the development of this procedure, perhaps with a microendoscope. The minimal trauma to the eye of this procedure leaves all other options of surgery open.

Laser-assisted patch clamping: a methodology.

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Adenovirus-Mediated Hepatic Gene Transfer in Mice: Comparison of Intravascular and Biliary Administration

Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of their ability to transduce many cell types with high efficiencies in vivo. After intravenous infusion, the majority of the vector is found in hepatocytes, but vector DNA is found to varying degrees in other tissues. In an attempt to restrict adenovirus-mediated gene transfer to the liver, we developed a microsurgical method that allowed for vector administration directly into the biliary tract of a mouse. We demonstrate that gene transfer was 4- to 10-fold more restricted to the liver after biliary tract infusion than after intravascular infusion. Intravascular infusion of recombinant adenovirus elicits a powerful immune response that limits gene expression and the ability to readminister the vector. Biliary infusion resulted in a slightly lesser immune response as determined by the lower neutralizing antibody titers directed against the vector compared with animals treated by intravascular infusion. There was no difference in the persistence of gene expression, suggesting a similar cell-mediated immune response against the vector containing cells in animals administered vector by either method. As future-generation adenovirus vectors that are safer and less immunogenic become available, the more liver specific gene transfer via the biliary tract may offer advantages over intravenous infusion for hepatic gene therapy.

Microsurgical Vasovasostomy

ABSTRACT: Over 3% of patients who have had a vasectomy now ask for reversal of their sterility in the United Kingdom, with divorce rates of more than one in three. Patency rates following vaso/asostomy using the usual methods with either no magnification or a loupe commonly result in patency rates of about 60–65% with ensuing fertility in about half of these cases. The thick‐walled vas has a lumen diameter of less than 1 mm, and there is often considerable disparity between proximal and distal ends resulting in obstruction as a major cause of failure. Division in the convoluted part of the vas, which is not uncommon, makes anastomosis even more difficult. Only accurate microsurgical anastomosis will consistently create optimal conditions for continuity without stenosis or leakage.The present paper describes the technique used and outcome of patency and fertility obtained in a personal series of the first 103 cases of microsurgical vasovasostomy, employing a two‐layer anastomosis carried out over a 5‐year period between 1 and 6 years ago. Ten had had a failed reversal operation elsewhere. A two‐layer microsurgical method of end‐to‐end anastomosis was employed, using a fine intraluminal nylon splint as a guide, which was withdrawn before the last interrupted suture was tied. Initially 8/0 Vicryl was used, and subsequently, with more experience, 10/0 Ethilon was employed.Sperm in the ejaculate was achieved in the whole series of bilateral primary operations in 85% rising to 94% with the use of 10/0 suture material and more experience. The patency results obtained match well those of previous microsurgical series, the largest recent one of Belker et al (1991) reporting 86% patency from five institutions, similar to whether a two‐layer or a modified one‐layer anastomosis was employed. However, a two‐layer anastomosis with approximation of the mucosa first is likely to attain more accurate results, particularly in the presence of disparity between the distal and proximal lumina, which is the usual finding (Owen and Kapila, 1984; Silber, 1988). It is for this reason that the two‐layer method was employed, and the results have been very gratifying with the patency rates achieved.Sperm concentration, quality, and motility improved in half the patients over the next 12 months. Fertility over a range of 12 months to 6 years postoperatively was 64% if vasectomy had been performed less than 10 years before, and 39% if 10 years or more had elapsed. In patients with previously failed reversal attempts elsewhere, 40% attained sperm in the ejaculate and half of these became fertile.The additional time and effort required for a microsurgical approach to vasovasostomy were worthwhile to obtain these results.

Production of chimeric calves by aggregation of in vitro-fertilized bovine embryos without zonae pellucidae

Bovine embryos produced by in vitro maturation (IVM), fertilization (IVF) and culture (IVC) were used to produce aggregation chimeras. An aggregated chimera was produced by combining bovine IVF embryos (Holstein × Japanese Black and Japanese Brown × Limousin breeds) which were cultured in vitro without the zonae pellucidae. Forty-eight hours after IVF, embryos at the 8 cell-stage were used to produce aggregation chimeras. In Experiment I, the zonae pellucidae was removed by a microsurgical method using a microblade or by treatment with 0.25% pronase. Holstein × Japanese Black embryos were aggregated with Japanese Brown × Limousin embryos after zonae removal by hand manipulation in culture medium. In Experiment II, the viability of the aggregated embryos developing into blastocysts was examined by measuring the extent of development. The number of aggregated embryos and embryos developed into blastocysts was 34 (91.9%) and 24 (70.6%), respectively, when the zonae pellucidae was removed by the microsurgical method; and 12 (92.3%) and 6 (50.0%), respectively, when the zonae pellucidae was removed using the 0.25% pronase treatment. The size of the aggregated embryos was significantly different from that of the normal embryos when cultured in vitro until Day 10, but not different thereafter. Five aggregated embryos were transferred nonsurgically to the recipients, resulting in 1 pregnancy and the birth of 2 chimeric calves. Skin color was used as evidence of chimerism.

Results obtained from microsurgical therapy of varicocele.

The microsurgical method of varicocelectomy was performed in 56 patients. In 25 cases the procedure was done because of scrotal symptoms and in 31 cases as a treatment for infertility. Complete regression of varicosities was observed in all the cases including 2 recurrent ones. The conception rate was 56% in the infertility patients excluding azoospermic and severe oligozoospermic ones. This microsurgical method has a good success rate and is also quite satisfactory in terms of its pregnancy rate.

Laser Surgery for Cervical Intraepithelial Neoplasia

The laser has provided a relatively easy and safe method for treating all types of CIN. The advantages of lasers include great conservatism due to tissue sparing, great precision because of microsurgical method, combination of excisions and vaporization possible, suitable for therapy of multifocal disease, uncluttered field, and good hemostasis. Although other modalities have also been used successfully in the therapy of this disease, it appears that none are so versatile as CO2 laser or possess its ability to accurately treat the multifocal disease that may involve large surface areas of the lower reproductive tract. It seems unlikely that any of the cervical ablation methods--chemical destruction, hot cautery, diathermy electrode, cryoprobe, laser, and diathermy loop--will completely disappear from use in the near future. Ablation is an attractive alternative to cold-knife excision in properly triaged patients, since it is almost always an outpatient procedure done without anesthesia or with only local anesthesia. Most importantly, a large number of patients have completely visible lesions of a severity less than that of in situ cancer; they really do not need excisional conization by any technique and benefit by quick ablation of the transformation zone. A conization, to be diagnostic and therapeutic, must remove the entire transformation zone to the proper depth. This procedure is almost always attended by a higher morbidity rate than is simple ablation. Laser excisional conization and the large loop excision of the transformation zone procedure are similar in a number of respects, because the operator must have certain capabilities and a through understanding of the disease to be treated to perform the operation correctly.

Microvenous Grafts In Reconstructive Lymphatic Microsurgery: 7 Years' Clinical Results

The author reports his experience with 32 cases of lymphatic-venous-lym phatic anastomoses (LVLA), an alternative microsurgical solution, personally conceived, proposed for the treatment of chronic lymphedemas of limbs, partic ularly in those cases in which there exists a venous disease associated with the lymphatic one in the same limb and, therefore, in which the most common lymphatic-venous anastomoses (LVA) would not be indicated. The preliminary diagnostic investigations, including lymphoscintigraphy, lymphography, and above all, the venous flowmetry and manometry be Doppler's method, play an important role in a precise choice of the indications for this kind of microsurgical technique, for they allow the investigator to point out, besides the lymphatic stasis, a more or less disguised venous pathology. The author's microsurgical method consists in the interposition of venous grafts, more or less long and with a suitable caliber, between lymphatic vessels placed upon and below the obstacle to the lymphatic circulation of the limb. The results of this personal LVLA microsurgical technique proved to be very satisfactory. All 32 cases treated by this method showed a significant and perma nent decrease of the edema, with a follow-up till five years after operation.

MODEL OF ORTHOTOPIC RENAL TRANSPLANTATION IN THE RABBIT

A microsurgical method of orthotopic renal allotransplantation in the rabbit is described, and the results of 179 consecutive procedures are presented. There were 10 (5.5%) deaths within 1 week of transplantation. Early complications included vascular thrombosis (4.4%), and obstruction (6.7%) and fistula formation (0.6%) at the uretero-ureteric anastomosis. This method of end-to-end vascular and ureteric anastomoses is straightforward and, in contrast to other models, does not require occlusion of the recipient inferior vena cava or aorta, use of a long segment of donor ureter, or operation on the recipient bladder.

Regeneration des Cortex bei Stentor coeruleus / Regeneration of the Cortex in Stentor coeruleus

Treatment of Stentor with very acid culture medium (pH 2.8) results in delamination of the cortex from the endoplasm . This procures the possibility to strip off the cortex with all its acid resistant organelles at minimum loss of endoplasm. Acid sensitive organelles react with dissolution or more or less damage. The endoplast may survive giving rise to a thin plasma membrane, which may be (even several times) shed and renewed. 10.5% of the decorticated cells regenerated a complete cortex. The ball-shaped and apparently apolar endoplast may pass through an ameboid stage. The visible part of the regeneration process begins with the appearance of a chaotic line system , which in regaining the polar order turns out to be the pigment free interstripe spaces. The ubiquitous appearing of foot anlagen underline the apolar situation. Nevertheless, the problem of endoplasmic preservation of polarity requires further, presently running, experiments. Obvious polarity begins to restore with the development of first membranelies and - approximately in opposite position with these - the definitive foot develops with the holdfast organelle. The ordering of stripe pattern starts from the foot and proceeds to the cells apex. With respect to Tartar’s contradicting result (death o fall decorticated cells) the new experiments do not refute Tartars conclusions, since the technique is different from his only micro-surgical method. This will be discussed in a following paper in context with the results of some other experiments. The here described total regeneration of the cortex in Stentor is not consummated as cytotactic differentiation depending on preformed structure.

Development of myoplasm-enriched ascidian embryos

The fertilized ascidian egg is thought to be comprised of distinct regions of tissue-specific cytoplasmic determinants. This idea was tested by bisecting fertilized eggs into egg fragments and culturing them until the unoperated controls developed into larvae. Fertilized eggs were bisected using a microsurgical method in which part of the uncleaved zygote was extruded through a hole made in the follicular envelope and the cytoplasmic bridge between the two egg regions was severed. One egg fragment contained all of the egg myoplasm (termed myoplasm-enriched or ME fragment), while the other fragment lacked myoplasm. ME fragments consisting of 40–50% of the total egg volume in many cases cleaved normally and developed into larvae. In a few cases, ME larvae initiated metamorphosis and developed into normal juveniles. Triton-extraction of ME embryos and larvae showed that the myoplasm was redistributed into nonmuscle lineage cells at each stage of development. Despite the redistribution of myoplasm into many of the endoderm cells situated in the head region of ME larvae, the expression of the muscle-specific enzyme acetylcholinesterase (AchE) and a muscle-specific antigen (Mu-2) was restricted to the tail muscle cells. The endoderm cells situated in the head region of ME larvae expressed an endoderm-specific enzyme alkaline phosphatase (AP) as in the controls. Furthermore, cleavage-arrested four- and eight-cell ME embryos expressed AchE activity in the expected number of blastomeres. When a greater quantity of myoplasm was redistributed into cells that normally do not express AchE activity by producing 10–30% ME embryos, in a few cases more than the expected number of blastomeres expressed AchE activity. In conclusion, the main finding of the present investigation, based on the development of ME fragments comprising 40–50% of the total egg volume, is that ascidian embryos are capable of regulative development.

Localization of axial determinants in the vegetal pole region of ascidian eggs

We have investigated the possibility that factors involved in embryonic axis determination are localized in the cytoplasm of ascidian eggs. Specific cytoplasmic deletions were carried out using a microsurgical method which consisted of extruding a portion of the egg or uncleaved zygote through a hole in the follicular envelope and severing it from the remaining ooplasm. When 5–15% of the total cytoplasm was deleted from the vegetal pole region of zygotes between the first and second phase of ooplasmic segregation, the vegetal pole-deficient embryos cleaved normally but did not gastrulate or develop into tadpole larvae. Instead, they formed radialized blastulae consisting of several hundred cells and with no apparent embryonic axis. The vegetal pole-deficient embryos express the muscle-specific enzyme acetylcholinesterase (AchE) and the endoderm-specific enzyme alkaline phosphatase (AP) in the expected number of blastomeres when cleavage arrested at the 8-cell stage, however. In contrast, normal larvae were usually obtained if similar or greater amounts of cytoplasm were removed from the vegetal pole region of unfertilized eggs, from the animal pole or equatorial regions of zygotes between the first and second phase of ooplasmic segregation, or from other regions, including the vegetal pole region, of zygotes that had already completed the second phase of ooplasmic segregation. Fate mapping indicates that the vegetal pole region enters four vegetal blastomeres of the 64-cell embryo and these are the first cells to invaginate during gastrulation. These results suggest that factors specifying the site of gastrulation and embryonic axis determination are transiently localized in the vegetal pole region of the ascidian zygote.

Nonsuture microsurgical vessel anastomosis.

An alternative microsurgical method for achieving vessel union without applying sutures is presented. The basic principles of the technique involve the creation of an extraluminal cuff that permits exact adaptation of both endothelial layers, without intraluminal damage or application of foreign material. When used for microsurgical procedures, the method can be further simplified by using a Teflon cuff secured by means of a mini-clip. The anastomosis can be accomplished quickly, in an uncomplicated and precise fashion. On the basis of preliminary experiments, this method appears to offer a wide range of applications, particularly if absorbable material is used.

Microsurgical bisection of expanding blastocysts for sexing of pig embryos.

A microsurgical method which is available for sexing of pig embryo was developed. A total of 22 pig expanding blastocysts was subject to being bisected into two hemispheres by a glass needle: one was inner cell mass (ICM)-half which contained whole ICM and approximately half of trophoblast, and other was the trophoblast (Tr)-half which had remaining half of trophoblast. After microsurgery, pairs of ICM and Tr halves were cultured for 20 hr, and ICM halves were transplanted to recipient gilts and Tr halves were used for chromosome analysis. The rate of development of transplanted ICM halves were 63.2% (12/19). This is approximately equal to usual results obtained in transplantation of pig embryos. Sixteen (80%) of 20 chromosome specimens from Tr halves showed one or more metaphase plates. The average of cells per specimens was 65.8. This result indicated that micromanipulation method presented here is a suitable one for determining sex of blastocysts of pigs. Enough cells for specimen preparation can be collected without descreasing embryo survivability.

A microsurgical method for reconstruction of interrupted lymphatic pathways: autologous lymph-vessel transplantation for treatment of lymphedemas.

Refinements in microsurgery have made it possible to perform causal therapy on lymphedemas due to a local blockade of lymphatic pathways through transplantation of the patient's lymph collectors. End-to-end anastomoses with lymphatics before and after the blockade or crossing to the opposite side are performed under 40-fold magnification. Between July 1980 and February 1985 32 patients received this treatment. 23 patients had postmastectomy edema, and 9 patients had unilateral edema of the lower limb (2 primary, 7 secondary edemas). The lasting result was that the volume difference between the affected and the healthy limb decreased to about 65%. Lymphatic scintiscans showed improved lymphatic transport capacity with increasing time from surgery and long patency of the grafts.

A microsurgical method for experimental kidney denervation.

A successful microsurgical method for denervation of the kidney in the rat is presented. Unilateral kidney denervation was performed in eight animals. Ten sham-operated animals were used as controls. Under ether anaesthesia, the kidney vessels on one side were exposed. The peritoneum above the vessels was incised and the area was stained with 1% toluidine blue solution. The nerves were identified. With microsurgical technique, the nerves were carefully separated from the vessels and resected. Tissue specimens from both kidneys in all animals were removed and analysed for norepinephrine content. There was no normal difference between the kidneys in the sham-operated rats. About 1 week after denervation, the norepinephrine concentration in the denervated kidney showed a 95% reduction, indicating a complete denervation. The described microsurgical procedure was a rapid, simple, and reproducible method for kidney denervation in the rat.

A potential microneurosurgical method for the manipulation of the developing nervous system of the postimplantation mouse embryo in utero. I. Injection methods

A microsurgical method for the injection of cells into the postimplantation mouse embryo whilst it is in utero is described. Specific regions of the 9.5-day-old mouse embryo brain have been injected with marker substances, such regions including the intraventricular cavity of the telencephalon and the trigeminal facialis ganglion bulge of the forebrain. Histological study of the injected embryos shows that the injection method is precise and able to avoid damage to other nearby embryonic tissues. The method should have wide application in the study of questions of normal cell lineage, neoplasia, teratology, and toxicology.

Results of direct coagulation of the ciliary body and cyclodialysis in hemorrhagic glaucoma

This paper discusses direct coagulation of the ciliary body as a microsurgical method for treatment of hemorrhagic glaucoma. During a follow-up period of 6-12 months intraocular pressure was lowered in all cases from an average of 42.8 mm Hg. Of the 26 patients operated on, 25 were subsequently free of pain. Stabilization of visual acuity was achieved in two-thirds of the cases. The surgical procedure is simple and involves little risk.

Microsurgical method of reuniting ductus epididymis

Based upon the experience of microsurgical treatment of 24 patients (45 operations), we consider epididymitis to be the main cause of obstructive aspermia. Thirty-eight interventions have been performed on the occluded epididymides. A new method of seminal vias restoration evolved by us is a microsurgical epididymoneostomy. This method consists of creating an anastomosis between two parts of the ductus epididymis, bypassing its scarred focus, with or without resection of the latter. Twelve such operations were performed in six patients. Follow-up in four of the patients revealed sperm in all cases. Although fertilizing characteristics of the sperm thus obtained were low, they were better than those obtained by other methods, excluding vasovasostomy, the simplest operation providing very good results. In dealing with spermostasis of obscure genesis, we attach special importance to the "isthmic" tubule, i.e., the part of the ductus epididymis connecting its corpus and cauda. Shut-down of this tubule by shunting epididymo-epididymoanastomosis resulted in spermatozoa appearance in an ejaculate in two of three such patients. We recommend application of microsurgical technique in the treatment of obstructive aspermia.

A Microsurgical Method for Denervation of the Liver in the Rat

The role of the nerves in the hepato-duodenal ligament should be of special surgical interest, since these nerves are often injured in hepatic and biliary tract surgery. We have developed a microsurgical procedure for liver denervation. All visible nerves were resected after staining. Norepinephrine values in central liver tissue of denervated rats were only 18% of the corresponding values in innervated liver tissue, indicating an appreciable degree of sympathetic denervation. No detectable immunoreactivity of gastrin, cholecystokinin, vasoactive intestinal polypeptide, substance P, somatostatin or enkephalin were found within resected hepatic nerves. The described denervation procedure is simple and reproducible. It can be used to obtain additional information about the nervous regulation of various liver functions.