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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.In the present work, Lister Hooded rats of both genders were used, aged 2-3 months and weighing 180-320 g. However, the procedure can be adapted in different rat strains of various age ranges.Ocular hypertension surgery and clinical follow-up<ol><li>House animals in a controlled temperature environment and 12 h light/dark cycle (6 am: light on/ 6 pm: light off) with standard pelletized gamma-irradiated food (Nuvilab&#174; CR-1, Quimtia S/A, Brazil) and water (triple filter, with non-toxic activated charcoal) available&#160;ad libitum.</li><li>Prepare a stock anesthetic cocktail mixture composed of three parts of 10% ketamine hydrochloride and one part of 2% xylazine hydrochloride (diluent: sterile water).</li><li>Gently restrain the animal by holding the dorsal skin and induce anesthesia by&#160;intraperitoneal administration of 1 &#181;L/g of body weight of the mixture (ketamine: 75 mg/kg; xylazine: 5 mg/kg). Check for proper anesthetization after approximately 5 min by performing a toe pinch response.</li><li>Topically anesthetize both eye surfaces by instilling proxymetacaine hydrochloride 0.5% eye drop. Wait for 30-60 s and remove the remaining solution from the anterior aspect of the globe, by gently touching the nasal or lateral bulbar conjunctiva with a small sterile cotton swab.</li><li>Measure baseline IOP of both experimental and contralateral control eyes by placing the animal in a ventral decubitus position on the benchtop such that the corneal surface is easily accessible to the tip of the tonometer.</li><li>Use either applanation or rebound handheld tonometer (Figure 1A). Position the tonometer tip such that it slightly touches the central corneal zone perpendicularly. Acquire and average 3-5 reliable machine-generated averages. Each mean is automatically calculated by the device after six successful individual measurements.<ol style='list-style-type:decimal;'><li>Load the rebound tonometer with a probe and press the measurement button once to turn it on, while placing the tip of the device upwards to avoid the probe falling. After turning it on, the display will show&#160;00&#160;indicating that the equipment is ready to measure.</li><li>Position the device with the tip of the probe at 1-4 mm from the cornea and acquire measurements by rapidly and carefully pressing the measurement button, without moving the equipment. Each successful measure is identified by a short beep and after six times, the mean is displayed on the device's screen. NOTE: Despite a long experience with applanation tonometer, to optimize the entire procedure the authors recommend the use of a rebound tonometer, which provides easier acquisition of reliable IOP measurement.</li></ol></li><li>Place the animal on a slight lateral decubitus position under a stereo microscope, and carefully plan the surgery by inspecting the experimental eye at 40x magnification. Do not forget to maintain the contralateral control eye lubricated during the procedure by instilling a drop of carmellose sodium or sodium hyaluronate.</li><li>With the aid of curved forceps, gently push forward the experimental eyeball so as to expose the vasculature that surrounds 360&#176; of the limbus. With the other hand, gently cauterize the vessels all around the cornea with a low-temperature ophthalmic cautery (1,300 &#176;F; Bovie Medical, USA) (Figure 1B-E). NOTE: The mentioned cautery has a round tip that should touch limbal vasculature longitudinally.</li><li>Be careful not to cauterize the corneal periphery, as this may result in post-operative corneal opacification, which precludes&#160;in vivo&#160;assessment of retinal function.</li><li>Observe the emergence of small circular marks of cauterization on the scleral limbus, the obliteration of limbal vasculature, and pupil dilation in the operated eye, which are signs of a successful surgical procedure. NOTE: As the limbal vasculature of rats and mice is anatomically similar&#160;and the cautery used has a gentle small tip, this protocol can work for the mouse eye as well without any adaptation.</li><li>After surgery, check the immediate post-operative IOP in both eyes such as described in step 1.5.</li><li>Apply a drop of ophthalmic prednisolone acetate (1.2 mg/mL) and maintain this in contact with the anterior surface of the experimental eye for around 40 s, then replace it with an ophthalmic ointment of antibiotics (oxytetracycline hydrochloride 30 mg/g plus polymyxin B 10,000 U/g; or ciprofloxacin 3.5 mg/g). Perform intramuscular injection of tramadol hydrochloride (single dose; 2 mg/kg) to prevent pain after the procedure.</li></ol>

<figure class='table' style='width:872pt;'><table class='ck-table-resized'><colgroup><col style='width:21.9%;'><col style='width:28.33%;'><col style='width:12.96%;'><col style='width:36.81%;'></colgroup><tbody><tr><td style='height:14.5pt;'>Cautery Low Temp Fine Tip 10/bx</td><td>Bovie Medical Corporation</td><td>AA00</td><td>Low-temperature ophthalmic cautery</td></tr><tr><td style='height:14.5pt;'>Cetamin</td><td>Syntec do Brasil Ltda</td><td>000200-3-000003</td><td>Ketamine hydrochloride 10%</td></tr><tr><td style='height:24.25pt;'>Ecofilm</td><td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td><td>MS-1.0298.0487</td><td>Carmellose sodium 0.5%</td></tr><tr><td style='height:24.25pt;'>Hyabak</td><td>Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A</td><td>MS-8042140002</td><td>Sodium hyaluronate 0.15%</td></tr><tr><td style='height:14.5pt;'>Icare Tonolab</td><td>Icare Finland Oy</td><td>TV02 (model number)</td><td>Rebound handheld tonometer</td></tr><tr><td style='height:24.25pt;'>Maxiflox</td><td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td><td>MS-1.0298.0489</td><td>Ciprofloxacin 3.5 mg/g</td></tr><tr><td style='height:24.25pt;'>Ster MD</td><td>Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A</td><td>MS-1.0497.1287</td><td>Prednisolone acetate 0.12%</td></tr><tr><td style='height:24.25pt;'>Terolac</td><td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td><td>MS-1.0497.1286</td><td>Ketorolac trometamol 0.5%</td></tr><tr><td style='height:24.25pt;'>Terramicina</td><td>Laborat&#243;rios Pfizer Ltda</td><td>MS-1.0216.0024</td><td>Oxytetracycline hydrochloride 30 mg/ g + polymyxin B 10,000 U/g</td></tr><tr><td style='height:24.25pt;'>Tono-Pen XL</td><td>Reichert Technologies</td><td>230635</td><td>Digital applanation handheld tonometer</td></tr><tr><td style='height:24.25pt;'>&#160;</td><td>Carl Zeiss</td><td>-</td><td>Stereo microscope for surgery and retinal dissection</td></tr></tbody></table></figure>

 induction of elevated intraocular pressure in a rat model

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)</a>This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

<img src='https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/23307/23307_Figure1_editor_23307.jpg' alt='23307_Figure1.jpg' />Figure 1: Thermal cauterization of limbal vascular plexus and consequences to retinal function&#160;in vivo.&#160;(A) Alternative methods to measure IOP in rats: rebound tonometry (superior), and applanation tonometry (inferior). (B-E) Surgical procedure; arrowhead: limbal vascular plexus; arrow: curved forceps used to expose anterior surface of the eyeball and optimize surgical assessment to limbal vessels; hash: the round tip of the low-temperature ophthalmic cautery; asterisk: cauterization mark. Scale bar: 2 mm. Inset in (D) shows in higher magnification the limbal vasculature to be cauterized. (F) Time course of IOP measurements in OHT (red) and control (black) eyes (n = 131). Vertical downward arrow: LPC surgery. * = p &lt; 0.05 (G) Arena for optomotor response analysis, comprised of four computer monitors arranged in a quadrangle, with a platform in the middle. The monitors display the image of a virtual cylinder composed of vertical alternate black and white stripes moving around the animal with constant rotational speed and variable spatial frequencies. The red crosshair corresponds to the center of the virtual cylinder. (H) Optomotor responses. Two distinct phases are distinguished upon surgical follow-up: the OHT phase (0-5 days) and the secondary degeneration phase (6-30 days). **** = p &lt; 0.0001. (I) Electrodes and animal positioning for pattern-ERG (PERG) acquisition: the active electrode at the temporal periphery of the cornea, and the reference and ground electrodes into the subcutaneous tissue of the ipsilateral temporal canthus and one of the hind limbs, respectively. The animal is positioned 20 cm from the stimulus screen. (J) PERG amplitude upon stimuli with different spatial frequencies. Like optomotor response, PERG evaluation also shows two distinct phases of responses after surgery: ocular hypertensive at 3 days post-surgery, followed by recovery at day 7 and 14, although still statistically lower than na&#239;ve responses, and a subsequent decrease at day 30 after surgery. c/d = cycles per degree. Na&#239;ve group: eyes of animals unexposed to any previous experimental manipulation. (H) and (J) show mean &#177; SEM, plus individual replicates in (H).

 Induction of Elevated Intraocular Pressure in a Rat Model

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This ...

Take an anesthetized rat and measure the intraocular pressure, or IOP, in both the experimental and control eyes.The IOP reflects the balance between aqueous humor production by the ciliary bodies and its drainage through the limbal blood vessels located between the cornea and sclera.Under a microscope, clamp the experimental eye to expose the limbal blood vessels.Use an ophthalmic cautery, a heat-generating device, to seal the vessels around the corneal circumference.The appearance of circular marks and pupil dilation indicates successful cauterization.The heat coagulates the connective tissue proteins in the blood vessel walls, forming a plug that impairs the aqueous humor outflow and raises the IOP.Measure the post-operative IOP to assess the elevated pressure in the experimental eye compared to the control eye.

induction-of-elevated-intraocular-pressure-in-a-rat-model

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

6 vistas. Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022) This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison. 

Video:  Induction of Elevated Intraocular Pressure in a Rat Model - Concepto

Ultrasound Cyclo Plasty in Eyes with Glaucoma

Glaucoma is a chronic disease caused by the progressive degeneration of the optical nerve fibers, resulting in decreased visual field that can lead to severe visual impairment, and eventually blindness. This manuscript describes a simple, surgeon-friendly, non-incisional technique, named Ultrasound Cyclo Plasty (UCP), for reducing intraocular pressure (IOP) in glaucoma patients. The technique determines a selective coagulation necrosis of the ciliary body; in addition, the stimulation of supra-choroidal and trans-scleral portions of the uveo-scleral outflow pathway has been recently proposed. UCP shows several technical improvements in ultrasound technology compared to previous techniques, providing more precise focusing on the target zone. The procedure is performed in the operating room under peribulbar anesthesia. Briefly, the coupling cone is put in contact with the eye and the ring probe, that contains six piezoelectric transducers which produce the ultrasound beams, is inserted inside it. Their proper centering over the ocular surface represents a crucial step for the correct targeting of the ciliary body. Sterile balanced salt solution is used to fill the empty spaces to ensure ultrasound acoustic propagation. Surgical treatment consists in the sequential automatic activation of each of the six transducers, for a total duration of less than 3 min. The patient leaves the hospital 1 h after the procedure with the treated eye patched. In the present study, 10 patients with open-angle glaucoma were followed-up during at least 12 months after the procedure. IOP was reduced at each interval compared to pre-operative, as well as the number of hypotensive medications. Twenty percent of patients did not respond to the treatment, and needed subsequent surgery to better control IOP. Treatment tolerability was good, with no cases of hypotony or phthisis. The UCP procedure is simpler, faster, safer, and less invasive than traditional cyclodestructive procedures with similar results in reducing IOP.

Glaucoma is a chronic disease with progressive degeneration of optic nerve fibers resulting in decreased visual field. Elevated intraocular pressure is considered the most important and the only treatable risk factor. This manuscript describes a simple, surgeon-friendly, non-incisional technique, named Ultrasound Cyclo Plasty, for reducing intraocular pressure in glaucoma patients.

Ultrasonido Cyclo plastia en ojos con Glaucoma

Glaucoma is a chronic disease caused by the progressive degeneration of the optical nerve fibers, resulting in decreased visual field that can lead to ...

Investigación

JoVE Journal

Comportamiento

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Un modelo de Glaucoma inducido por sutura de Circumlimbal en ratas y ratones

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Medicina

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

Evaluación del glaucoma de ángulo abierto en etapa temprana en pacientes mediante un potencial evocado visual de comprobación aislada

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

Induction of Elevated Intraocular Pressure in a Rat Model

Transcripción

Induction of Elevated Intraocular Pressure in a Rat Model

Ultrasonido Cyclo plastia en ojos con Glaucoma

Un modelo de Glaucoma inducido por sutura de Circumlimbal en ratas y ratones

Evaluación del glaucoma de ángulo abierto en etapa temprana en pacientes mediante un potencial evocado visual de comprobación aislada