Laser Transection Research Articles

O252: Studying the neuronal regenerative response to in-vitro laser axotomy

Abstract Aims Outcomes of peripheral nerve injury (PNI) remain suboptimal, and the intrinsic mechanisms directing regeneration of injured peripheral neurons are not completely understood. To study the cellular response to injury, it is critical to deploy a model that facilitates a precise and controlled transection. We aim to recreate clinical outcomes at a micrometer level and analyse the impact of injury parameters, such as the proximity of injury to the cell body, on neuronal survival and regeneration. Methods We cultured adult rat dorsal root ganglion (DRG) neurons and transected individual neurites with a high-precision laser at 25um and 125um from the cell body. We monitored for 24 hours using time-lapse microscopy and analysed cell survival and neurite regeneration, comparing between two distance groups. Results Following laser transection at 25um from the cell body, 56% (n=25) of neurons survived for 24 hours and 33.3% (n=12) regenerated. When laser-transected at 125um, 75% (n=40) of neurons survived and 73.1% (n=26) regenerated. The mean regenerative distance was 19.9um (n=4) when injured at 25um and 173.9um (n=19) at 125um. Discussion Our laser axotomy method facilitated efficient and precise transection of individual neurites. Distal injuries generated significantly higher survival rates and regenerative length and rates compared to proximal injuries. These findings mimic clinical outcomes where distal injuries have greater recovery than proximal injuries, thereby demonstrating the efficacy of this model for investigating cellular mechanisms directing peripheral neuron regeneration. We envisage that this model will facilitate future studies aiming to accelerate peripheral neuron regeneration in a clinical context.

Active shrinkage protects neurons following axonal transection

Trauma, vascular events, or neurodegenerative processes can lead to axonal injury and eventual transection (axotomy). Neurons can survive axotomy, yet the underlying mechanisms are not fully understood. Excessive water entry into injured neurons poses a particular risk due to swelling and subsequent death. Using invitro and invivo neurotrauma model systems based on laser transection and surgical nerve cut, we demonstrated that axotomy triggers actomyosin contraction coupled with calpain activity. As a consequence, neurons shrink acutely to force water out through aquaporin channels preventing swelling and bursting. Inhibiting shrinkage increased the probability of neuronal cell death by about 3-fold. These studies reveal a previously unrecognized cytoprotective response mechanism to neurotrauma and offer a fresh perspective on pathophysiological processes in the nervous system.

Rhinological Consequences of Microsurgical Endonasal-Transsphenoidal Surgery for Pituitary Tumors.

Transsphenoidal surgery for sellar lesions may affect patency and function of the nasal airways, smell and sinonasal quality of life. Below is our report on otorhinolaryngological data garnered from patients undergoing endonasal transsphenoidal pituitary microsurgery. In a prospective study, 68 patients scheduled for transsphenoidal operations (32 female, 36 male, age 17-72 years) underwent otorhinolaryngological evaluation of their nasal morphology, a standardized smell test (sniffin' sticks) and rhinomanometry to analyse nasal breathing function preoperatively, 3-5 days postoperatively (without rhinomanometry), after 3-4 months and after 9 months. Immediately after surgery, a reduction in smell sensation was detected in almost all patients. Within 3 months, this impairment resolved in all cases except one. In 2 patients (3%) with preoperative anosmia, improvement of smell function to>6 out of 12 sniffin' sticks was observed. At final visit no patient was noted to have new anosmia. Within 3 months, the results of the rhinomanometry revealed that all patients except one, regained their preoperative nasal breathing function. In 6 patients (8.8%) an improvement in their nose breathing abilities compared to the preoperative state was found. Three patients (4.4%) underwent a LASER transection of mucosal synechiae. In one case with persistent nasal obstruction (1.5%), secondary septoplasty had to be performed. There was no case in which perforation of the nasal septum, nasal tip deflection, or saddle nose deformity was observed. Microsurgical resection of pituitary tumors via the endonasal transsphenoidal approach poses an acceptable risk with regards to sinonasal complications. The incidence of secondary rhinosurgical interventions is low. Standardized comparative studies between endoscopic and microsurgical transsphenoidal operations should be undertaken.

Third-Order Neurons in the Lateral Horn Enhance Bilateral Contrast of Odor Inputs Through Contralateral Inhibition in Drosophila.

The survival and reproduction of Drosophila melanogaster depends heavily on its ability to determine the location of an odor source and either to move toward or away from it. Despite the very small spatial separation between the two antennae and the redundancy in sensory neuron projection to both sides of the brain, Drosophila can resolve the concentration gradient by comparing the signal strength between the two antennae. When an odor stimulates the antennae asymmetrically, ipsilateral projection neurons from the first olfactory center are more strongly excited compared to the contralateral ones. However, it remains elusive how higher-order neurons process such asymmetric or lateralized odor inputs. Here, we monitored and analyzed for the first time the activity patterns of a small cluster of third-order neurons (so-called ventrolateral protocerebrum neurons) to asymmetric olfactory stimulation using two-photon calcium imaging. Our data demonstrate that lateralized odors evoke distinct activation of these neurons in the left and right brain hemisphere as a result of contralateral inhibition. Moreover, using laser transection experiments we show that this contralateral inhibition is mediated by presynaptic neurons most likely located in the lateral horn. Finally, we propose that this inhibitory interaction between higher-order neurons facilitates odor lateralization and plays a crucial role in olfactory navigation behavior of Drosophila, a theory that needs to be experimentally addressed in future studies.

Corneal nerve healing after in situ laser nerve transection.

PurposeWe have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane.MethodsThy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption.ResultsThe pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence.ConclusionsInterconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection.

The ubiquitin ligase PHR promotes directional regrowth of spinal zebrafish axons

To reconnect with their synaptic targets, severed axons need to regrow robustly and directionally along the pre-lesional trajectory. While mechanisms directing axonal regrowth are poorly understood, several proteins direct developmental axon outgrowth, including the ubiquitin ligase PHR (Mycbp2). Invertebrate PHR also limits regrowth of injured axons, whereas its role in vertebrate axonal regrowth remains elusive. Here we took advantage of the high regrowth capacity of spinal zebrafish axons and observed robust and directional regrowth following laser transection of spinal Mauthner axons. We found that PHR directs regrowing axons along the pre-lesional trajectory and across the transection site. At the transection site, initial regrowth of wild-type axons was multidirectional. Over time, misdirected sprouts were corrected in a PHR-dependent manner. Ablation of cyfip2, known to promote F-actin-polymerization and pharmacological inhibition of JNK reduced misdirected regrowth of PHR-deficient axons, suggesting that PHR controls directional Mauthner axonal regrowth through cyfip2- and JNK-dependent pathways.

Оптимизация фемтосекундной лазерной транссекции гидрофобной интраокулярной линзы и отдаленные результаты в клиническом исследовании (первый опыт)

Purpose. To optimize the energy parameters of the FL assistance using the LenSx FL unit for transection of IOL made of hydrophobic material, as well as to develop a sparing surgical technique for the IOL removal from the anterior chamber. Material and methods. In the experimental part of the study, the femtosecond laser transection of IOL was carried out using the LenSx unit in different energies (1, 2, 4, 6, 8, 10µJ). The subsequent mechanical separation of IOL was evaluated according to a 4-point scale. Single IOL fragments after transection (n=3) were studied under experimental conditions using the optical light microscopy in 40 and X100 magnifications The preexplantation FL transection was performed under clinical conditions in one patient. The Evaluation of FL transsection of IOL was carried out using the electron microscope with magnification x47 and х800. Results. Under experimental conditions, the pulse energy levels (2, 4, 6, 8, 10 µJ) showed the similar efficiency of IOL transection. The level of energy 1µJ – manual separation of the IOL along the line of transection was impossible. High energy loads (8, 10µJ) entailed a change in the surface of IOL of unknown nature. In clinical conditions the technique of FL transection of IOL showed its safety and efficiency. Conclusion. Hydrophobic IOLs can be fragmented using the femtosecond laser.

Laser-based single-axon transection for high-content axon injury and regeneration studies.

The investigation of the regenerative response of the neurons to axonal injury is essential to the development of new axoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that the ability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We show that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely induce damage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axon regeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectories and established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. The characterization of the motile response of axotomized RGC-5 cells showed that cells that were capable of repair or regrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells with long axons could not recover their injured axons, and such cells were much more motile. The platform we describe allows highly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.

Epidermal growth factor promotes oligodendrocyte process formation and regrowth after injury

Oligodendrocytes (OLs) form myelin within the central nervous system and are targets in numerous demyelinating diseases and injuries. OLs grown in culture maintain the developmental timetable which occurs in vivo and mature into cells with a relatively normal phenotype. In this study, cultured cells are used to test whether EGF can modulate process formation in OLs both before and after transection injury. EGF had no effect on the formation of new processes by OLs at any stage of development. To test the effect of EGF on process outgrowth after injury, mature OLs were selected and injured by laser transection of a single process, then imaged at 24-h intervals for 120 h. EGF promoted the recovery and regrowth of injured processes and also significantly increased outgrowth in uninjured processes. As well, it increased the number of new sprouts formed by OLs after injury. Results suggest that the effects of EGF on process outgrowth are a consequence of EGF interaction with a signaling pathway that is specifically activated within injured OLs. The potent effect of EGF on OL process formation after an injury suggests that modulation of the signaling pathways involved might provide a mechanism to promote remyelination.

Epidermal growth factor promotes oligodendrocyte process formation and regrowth after injury

Oligodendrocytes (OLs) form myelin within the central nervous system and are targets in numerous demyelinating diseases and injuries. OLs grown in culture maintain the developmental timetable which occurs in vivo and mature into cells with a relatively normal phenotype. In this study, cultured cells are used to test whether EGF can modulate process formation in OLs both before and after transection injury. EGF had no effect on the formation of new processes by OLs at any stage of development. To test the effect of EGF on process outgrowth after injury, mature OLs were selected and injured by laser transection of a single process, then imaged at 24-h intervals for 120 h. EGF promoted the recovery and regrowth of injured processes and also significantly increased outgrowth in uninjured processes. As well, it increased the number of new sprouts formed by OLs after injury. Results suggest that the effects of EGF on process outgrowth are a consequence of EGF interaction with a signaling pathway that is specifically activated within injured OLs. The potent effect of EGF on OL process formation after an injury suggests that modulation of the signaling pathways involved might provide a mechanism to promote remyelination.

A new instrument for hemostatic vascular management in endoscopic surgery: diode laser (STATLase-SDL) and its handpiece (dual hook).

The authors described experimental data in hemostatic vascular transection using a STATLase-SDL with a 780-865 nm GaA/As diode wavelength and its handpiece, Dual Hook (DH). The STATLase-SDL and DH are newly developed devices for endoscopic surgery. It seems that the device has improved hemostatic cutting ability. The DH was applied to the abdominal aorta (< 2 mm) of Japanese white rabbits (n = 15) under general anesthesia. Tissue temperature during laser transection and interluminal bursting pressures were measured. Histopathological examinations at the cut end were carried out by hematoxylin-eosin (H&E) and elastica Van Gleson's (EVG) stainings. To investigate short-term effects 11 days postoperatively, the cut ends of the vessels were examined histologically in the heminephrectomy group. The DH hemostatistically cut through arteries up to 2 mm in diameter. Temperatures at the inner hook and in adjunct tissue were higher than 160 degrees C, while that at the outer hook was lower than 80 degrees C. The bursting pressures immediately after transection was 307.2 +/- 35.2 mm Hg at a laser power of 8 W (n = 5), and 295.6 +/- 28.7 mm Hg at 10 W (n = 5). Histological examination of the transected sites on the vessels revealed well-opposed tissue welding of the vascular wall. Absence of fragmentation in the welding of the internal elastic lamina is crucial for closure of the vessel stump. Up to 11 days postoperatively, there were no direct complications related to the use of the DH. The STATLase-SDL and DH have good hemostatic cutting ability and might be suitable for endoscopic surgery.

Effect of a modified Nd:YAG laser technique on neuroma formation: An experimental study in rat sciatic nerve.

Traumatic transection of a peripheral nerve is inherently associated with the development of neuroma at the end of the proximal stump, often leading to therapy-resistant pain. This study was designed to evaluate whether the neodymium:yttrium aluminum garnet (Nd:YAG) laser could prevent neuroma formation after neurectomy. The sciatic nerves of 14 rats were diffuse coagulated by defocused Nd:YAG laser (12 W power), and subsequently transected with additional focused laser energy. The control group consisted of contralateral nerves transected by microscissors. The nerves were reexposed at different time intervals up to 9 weeks after surgery, and evaluation consisted of macroscopy, and light and transmission electron microscopy. True neuroma formation could not be observed after laser transection, and only five nerves formed a neuromatous bulb, with minimal adhesions to surrounding tissue. Microscissor transection resulted in widespread amputation neuromas, consisting of regenerating axons and connective tissue, and nervous tissue regenerating into surrounding tissue. Laser-transected nerves showed degenerative changes of the axons and myelin, while proliferation of Schwann cells could not be observed. No outgrowth of axons could be observed outside the coagulated proximal stump. An epi/perineurial layer was present, covering the nerve stumps. Microscissor-transected nerves showed proliferation of fibroblasts and Schwann cells, forming minifascicles, and vigorous outgrowth of axons into the tissue and even into the distal nerve stump. Within the limitations of this study it is concluded that the formation of amputation neuromas is suppressed by Nd:YAG laser application by thermal coagulation of the nerve and suppression of Schwann-cell proliferation.

Real-time observation of the disassembly of stable neuritic microtubules induced by laser transection: possible mechanisms of microtubule stabilization in neurites.

By dissolving the membrane with detergent perfusion, we have shown that the established neurites of dorsal root ganglion cells cultured for more than 5 days contained microtubules which persisted outside the cell for a few minutes to more than 1h [Tashiro et al., 1997: J. Neurosci. Res. 50:81-93]. To investigate their stabilization mechanism, we transected the exposed microtubules by laser microbeam irradiation and observed their length changes with video-enhanced differential interference contrast microscopy. Microtubule fragments started to shorten on both sides of the transection site. more rapidly from the newly generated plus ends than from the minus ends. The maximal rate as well as the pattern of shortening correlated with the time of transection; microtubules transected later than 30 min after membrane removal shortened at rates less than 20 microm/min and typically with intermittent pauses, while the more labile microtubules included in the earlier transections shortened continuously at higher rates. Microtubules in neurites were thus stabilized by 1) stopping disassembly at local sites including the plus ends, and 2) slowing disassembly along the length. Transection also suggested the presence of specialized points along microtubules which are involved in anchoring microtubules to the substratum. Cell Motil. Cytoskeleton 42:87-100, 1999.

Real‐time observation of the disassembly of stable neuritic microtubules induced by laser transection: Possible mechanisms of microtubule stabilization in neurites

By dissolving the membrane with detergent perfusion, we have shown that the established neurites of dorsal root ganglion cells cultured for more than 5 days contained microtubules which persisted outside the cell for a few minutes to more than 1 h [Tashiro et al., 1997: J. Neurosci. Res. 50:81–93]. To investigate their stabilization mechanism, we transected the exposed microtubules by laser microbeam irradiation and observed their length changes with video-enhanced differential interference contrast microscopy. Microtubule fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The maximal rate as well as the pattern of shortening correlated with the time of transection; microtubules transected later than 30 min after membrane removal shortened at rates less than 20 μm/min and typically with intermittent pauses, while the more labile microtubules included in the earlier transections shortened continuously at higher rates. Microtubules in neurites were thus stabilized by 1) stopping disassembly at local sites including the plus ends, and 2) slowing disassembly along the length. Transection also suggested the presence of specialized points along microtubules which are involved in anchoring microtubules to the substratum. Cell Motil. Cytoskeleton 42:87–100, 1999. © 1999 Wiley-Liss, Inc.

Comparative study of laser and scalpel nerve transections

Comparative study of laser and scalpel nerve transections

Salpingectomy via Laparoseopy: A New Surgical Approach

This study presents 100 consecutive cases of total salpingectomy performed via laparoscopy for indications of ruptured or recurrent ectopic pregnancy, hydrosalpinges, torsion of the fallopian tube, hematosalpinges, or extensive adhesions. A multiple abdominal puncture approach was used, and salpingectomy was accomplished by electrosurgical coagulation and laser transection of the isthmus, mesosalpinx, and tubo-ovarian ligaments using the CO2 laser. The fallopian tubes were removed from the pelvic cavity through one of the suprapubic punctures. The mean duration of the procedure was 22 minutes, and the mean duration of hospitalization after surgery 7.4 hours. No major intraoperative or postoperative complications were encountered. Laparoscopic salpingectomy appears to be a safe and relatively simple procedure associated with the advantages of outpatient surgery.

Comparative study of neuroma formation in the rat sciatic nerve after CO 2 laser and scalpel neurectomy in combination with milliwatt CO 2 laser “sealing”

Nerve transections of rat sciatic nerves were performed to compare scalpel neurectomy to CO 2 laser neurectomy. Additionally, the effect of milliwatt CO 2 laser application to the proximal nerve ending was studied. Animals were sacrificed at 1 to 9 weeks, and the neuromas were examined histologically and measured. Scalpel transections produced typical neuromas. Laser transections produced neuromas characterized by multinucleated giant cells and carbonaceous debris. There was no difference in the size of the neuromas produced by either method of transection. Milliwatt CO 2 laser application to the proximal stump did not affect the size or histology of resultant neuroma formation regardless of the method of transection.

Regeneration of peripheral nerves following Nd-YAG laser and scalpel transection—Experimental observations

Regeneration of 70 rat sciatic nerves was investigated following transection by a 1.06 μm neodymium-doped yttrium aluminium garnet (Nd-YAG) laser and by a conventional scalpel. Twenty-three amputation stumps after each technique were reexposed at different intervals between 3 and 240 days. In an second series 12 nerve trunks each were taken out following initial epineural suture repair after survival periods between 30 and 90 days.

Prevention of neuroma formation by Neodym Yag laser--experimental observations.

The influence of Laser radiation on neuroma formation was investigated in rat sciatic nerves. Peripheral nerve trunks were divided by focused Neodym Yag Laser and compared with those transected by microscissors. The nerves were re-exposed at different time intervals up to 240 days after initial operation. True neuroma formation could not be observed after laser transection. Sharp division resulted in widespread amputation neuromas consisting of regenerating axons and connective tissue. Neodym Yag laser radiation by its scattering effect may cause deep coagulation necrosis and thrombosis of vasa nervorum thus sealing the dome of the divided nerve trunk. The formation of amputation neuromas may be suppressed by laser application.

Preliminary study of the Nd:YAG laser in canine partial nephrectomy.

Partial nephrectomy is effective in the treatment of segmental renal disease but hemostasis remains a challenge. In this preliminary investigation the Nd:YAG laser was used alone or as an adjunct to the scalpel in partial nephrectomies to determine if hemostasis could be improved. A technique of 100-W laser transection with occlusion of the renal artery was effective for partial nephrectomy and achieved complete hemostasis. Conditions of patent renal artery flow or renal cooling were associated with a reduction in necrosis depth with 100-W laser partial nephrectomies. All techniques were compatible with survival over the 5-day study period.