Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Summary

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Abstract

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca²⁺ signaling using two-photon microscopy.

Introduction

Traumatic spinal cord injury (SCI) is a devastating clinical condition affecting the sensorimotor and autonomic functions of the SC. Patients surviving SCI are often left with debilitating paraplegia that significantly affects their daily activities and quality of life¹. Experimental SCI models have been an indispensable tool in the scientific investigation to understand the pathophysiology of SCI and associated neural repair processes. These models have also been used to test the preclinical efficacy of various experimental neuroprotective interventions that are aimed at functional recovery. Currently, majority of the SCI models in practice employ the use of physical blunt force to mechanically disrupt and injure the SC. These methods include contusion, compression, dislocation and transection of the SC². It has been suggested that after the primary mechanical insult a secondary injury in the form of ischemia sets in in the injured SC^3,4. The etiology of secondary ischemia includes extensive tissue degeneration, parenchymal hemorrhage and sometimes by obstruction of blood vessels by tissue edema^5-7. As a result of the secondary injury the integrity of SC is further affected, neurons and glial cells are severely impaired in function and viability and undergo apoptosis which leads to infarct growth during the chronic stage of injury, analogous to the growth of ischemic penumbra following stroke^8,9. Several mechanisms like excitotoxicity, free radical production, and inflammation have been reported to be responsible for the ischemic cell death following SCI^10,11. In addition, SC ischemia is a serious complication of thoraco-abdominal aortic aneurysm repair surgeries which often lead to paraplegia in the patients^12,13. In spite of such high clinical impact very few models of spinal cord ischemia with high reproducibility are currently available.

Photothrombosis (PT) is a commonly used method for the induction of focal ischemia in the brain^14-20. The technique is fairly non-invasive, highly reproducible and produces a precise focal ischemic lesion in the exposed area of the brain^17-21. This is achieved by systemic administration of photoactive dyes like Rose Bengal (RB)^16-20,22 or erythrosine B²³ followed by localized irradiation of blood vessels with proper light source. Photoactivation of the dye causes the generation of free radicals which disrupt the integrity of the smooth vascular endothelium, and cause the platelets to accumulate, which subsequently forms a thrombus. The obstruction of blood flow by the thrombus results in an infarct in the region supplied by the vessel²⁴. Due to ease of control on the intensity and duration of irradiation this procedure yields a highly uniform and reproducible infarct. Furthermore, this method can be employed to induce an infarct at various anatomical locations enabling spatial (e.g., grey matter vs. white matter) understanding of the effect of ischemia.

The aim of the current study is to develop an easy and highly reproducible model of SC ischemia in mice. We described the procedure of a PT model of SC ischemia in mice. Results from histology and immunostaining demonstrated that PT can effectively induce SC infarction, neuronal loss and reactive gliosis.

Protocol

Note: Mice (C57BL/6J, male) aged 10 – 12 weeks were used in this study. All the procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Missouri Institutional Animal Care and Use Committee (IACUC). 

1. Pre-Surgery

1. The day before surgery autoclave and sterilize all the surgical instruments. Wrap the instruments and autoclave at 121 ^oC at 15 psi for 30 min followed by 30 min of drying (121 ^oC, 15 psi, 30/30 cycle). Place the instruments in a clean and sterile environment until further use.
2. Prepare fresh Rose Bengal (RB) solution (20 mg/ml in sterile saline) every time before the surgery. To completely dissolve RB, vortex the tube then followed by sonication for 5 min at 50/60 Hz with output power of 19 W. Wrap the tube in aluminum foil and protect it from light until further used during surgery.
3. Prepare a mixture of ketamine/xylazine in sterile saline. Add 125 µl of xylazine (stock concentration: 20 mg/ml) and 325 µl of ketmaine (stock concentration: 100 mg/ml) and 550 µl of sterile saline to make a final volume of 1 ml of anesthetic mixture.
4. Pre-warm the homeothermic heating pad.
5. Pre-warm the metal halide lamp (light source for FN1 epi-fluorescence microscope) for 30 min to stabilize the lamp power.
6. Adjust the size of the illuminated region to a diameter of 1 mm using 10X objective and a reticle by adjusting the field diaphragm in the upright FN1 epi-fluorescence microscope.

2. Surgical Procedure

1. Anesthetize the mouse with a dose of ketamine (130 mg/kg B. wt.) and xylazine (10 mg/kg B. wt.). Based on the cocktail in 1.3,  4 µl per g of mouse B. wt. are needed. Sterilize the site of injection with an alcohol swab and administer the anesthetics through the intra-peritoneal (IP) route. Take care not to inject the anesthetics into the blood vessels or muscle as this would delay the induction and recovery of the animal.
2. Apply artificial tear ointment to both eyes of the mouse to prevent drying and place the animal on the heating pad to prevent hypothermia.
3. Preparation of animal
   1. Check the animal for proper surgical level anesthesia by using the toe pinch response.
   2. Once the animal has reached surgical level of anesthesia, clip the hair on the dorsal surface around midline of the animal using an electric hair trimmer. Scrub the surgical site with 70% ethanol followed by betadine solution three times. Cover the site with a sterile surgical drape until next step.
4. Surgical procedure to thin the bone to expose the spinal cord
   1. Place the mouse in a prone position over the homeothermic heating pad on the surgical platform (Figure 1A). Properly secure the mouse posture using a snout clamp to maintain an elongated neck region (Figure 1A, B).
   2. Make an incision (approximately 1 cm long) using surgical scissors along the dorsal mid line extending from the thoracic vertebrae T9 to T12. Move aside the skin to expose surgical area.
   3. Using a scalpel, carefully clear the muscle to expose the dorsal spines at T9 - T12 vertebrae. Stop bleeding in each step by applying gentle pressure with sterile cotton swab. Separate T10 - T12 vertebrae from the surrounding muscle and secure them using a vertebral clamp to stabilize and prevent any movement (Figure 1A, B).
   4. Using a high speed drill with bone polishing drill bit, carefully and gently thin the dorsal surface of T10 or T11 vertebra to visualize the posterior spinal vein and other small vessels on the dorsal surface of the SC (Figure 1C).
   5. To prevent thermal damage due to heat generated during the thinning procedure, apply a gentle and constant stream of normal saline along with constant suction to remove the debris.
   6. Using a scalpel carefully smooth the bone surface until the main vessel is clearly visible. Take care not to damage the spinal cord during this process.
   7. Once the blood vessel is visualized, administer RB at a dose of 30 mg/kg (body weight) through retro-orbital sinus route using an insulin syringe.
   8. Measure blood flow using a Laser Doppler flowmeter after 3 min following RB injection if necessary (Figure 2A, B). Maintain asepsis during entire procedure.

3. Induction of PT

1. Place the animal on an X-Y position adjustable stage over a Lab-Jack which can adjust the height. Adjust the position of the mouse so the exposed region of T11 spinal cord is directly under the 10x objective of the FN1 epi-fluorescence microscope (Figure 3A).
2. Set the power of the light source at 12% and irradiate the T11 region with a diameter of 0.75 mm in the middle of thinned spinal cord (Note: this region includes the posterior spinal vein and other capillaries) with a green light (wavelength 540 - 580 nm, which is achieved by the filter cube in the microscope) through the 10X objective for 2 min. Take images at the beginning and end of irradiation (Figure 3B, C) and record the time of the experiment at this point.
3. Measure blood flow again for 10 min if necessary by placing the laser Doppler probe to the same position above the spinal cord as in 2.4.8 (Figure 2A, B).
4. After irradiation check for any hemorrhage and if none found proceed to suturing the animal. Suture the superficial fascia along with the muscles on the either side of spinal cord using an absorbable suture or 4-0 size silk suture. Take care not to damage the exposed SC. Suture the skin with 4-0 silk suture. Apply Betadine or iodine to the edges of the skin after suturing.

4. Post-surgery Care

1. After suturing, place the animal on the heating pad for recovery. After recovery check the animals for signs of neurological deficits by observing the movement of both hind-limbs. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.
2. Transfer the animals to the home cage. Do not return the animal that has undergone surgery to the company of other animals until fully recovered.
3. Check the animals at regular intervals. In case of severe neurological deficits, provide proper care like evacuation of bladder, administration of analgesics (buprenorphine, 0.05 - 0.1 mg/kg). Check for dehydration and administer normal saline subcutaneously in severe case. Usually, buprenorphine ( 0.1 mg/kg) will be administered after suturing to relieve pain in the surgical site.
4. If animals are not immediately euthanized after surgery, we will put high water content diet on the cage floor, so the animals can reach the food easily.

5. Transcardial Perfusion, Nissl staining and Immunostaining

1. Transcardially perfuse the animal as described previously^17-20.
   1. Anesthetize the animal as described earlier in the protocol and transcardially perfuse with phosphate buffer saline (PBS, pH 7.4), followed by ice-cold 4% paraformaldehyde (PFA) in PBS.
   2. After perfusion, remove the spinal cord (SC) and post-fix it in 4% PFA in PBS at 4 °C O/N. Transfer the fixed SC into PBS with 30% sucrose and keep it for 2 - 3 days until it sinks to the bottom of the tube.
   3. Using a cryostat cut the spinal cord into 30 µm thick sections and place them serially on a gelatin-coated glass slides or in a 48-well plate with 0.01 M PBS.
2. Nissl staining: To inspect the injury caused by PT perform a Nissl staining on spinal cord sections as previously described^17-20.
   1. Briefly, collect every fifth spinal cord slice on the glass slides and stain with 0.25% cresyl violet. Take images of the stained sections (Figure 4).
3. Immunostaining: As described previously using a floating section method^17,18,20.
   1. Briefly, stain the spinal cord sections by incubating O/N at 4 °C with rabbit anti-glia fibrillary acidic protein (GFAP) polyclonal antibody (1:300), rabbit anti-NeuN antibody (1:300), and rabbit anti-Iba1 antibody (1:500) followed by donkey anti rabbit Alexa 568-conjugated IgG (1:400) secondary antibodies for 4 h at RT. Take images with a fluorescent microscope (Figure 5).

Results

The aim of this study was to produce spinal cord ischemia in mice using a PT model. After the desired region of bone above the spinal cord (T10 - T12) was thinned, Rose Bengal was injected through retro-orbital sinus route, and ischemia was induced by PT. Figure 1A, B show the mouse positioned in a custom-made surgical platform during surgery. The mouse was held in place by a snout clamp and two adjustable vertebrate clamps to stabilize the spinal cord. Figure 1C show a thinned window ab...

Discussion

In this study, we described a photothrombotic model of SC ischemia. Due to advances in genetic engineering there has been a surge in commercially available transgenic mice which has made it possible to study the impact of specific genes involved in the ischemic pathophysiology in the SC. The aim of the study was to develop a reproducible mouse model of spinal cord ischemia. Here we adapted a cortical PT model to induce SCI in mice. Following surgery the posterior spinal vein and capillaries on the dorsal...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rose Bengal	Sigma-Aldrich	330000	20 mg/ml in sterile saline
C57BL/6J	Jackson lab	664	22 - 25 g
Ketamine	VEDCO	NDC-50989-996-06	100 mg/ml
Xylazine	VEDCO	NDC-50989-234-11	100 mg/ml
Betadine solution	Purdue	NDC-67618-150-01	10% povidone iodine topical solution
Normal saline	Abott Laboratories	04930-04-10	For diluting RB, anaesthesia and for preventing tissue from drying
Artificial tears ointment	Rugby	NDC-0536-6550-91	83% white petrolatum
Ethanol	Decon labs.Inc	2716	70% ethanol for disinfection
Metal halide lamp	EXFO, Canada	X-Cite 120 PC	Set power at 12%
Spring scissors	Fine Science Tool	15000-10	for minor dissection
Scissors (angled to side)	Fine Science Tool	14063-011	No. 3 handle
Standard scalpel	Fine Science Tool	10003-12	for removing muscle
Scalpel blade	Feather	2976	No. 10
Forceps (curved)	Fine Science Tool	11150-10	for holding tissue
Forceps (straight)	Fine Science Tool	11151-10	for holding tissue
Needle holder	Fine Science Tool	12002-12	for suturing
Tissue adhesive glue	3M Vetbond	1469SB	to adhere to edges of the cut skin
Monofilament polypropylene	USSC Sutures	VP-521	Size = 4-0 (for fascia)
Perma-hand silk	Ethicon	683G	Size = 4-0 (for skin)
Micro drill	Roboz Surgical Instrument Co. Inc.	RS-6300	with bone polishing drill bit
Laser doppler flowmeter	Moor Instruments	moorVMS-LDF1	for monitoring change in blood flow
Heating pad	Fine Science Tool	21052-00	to prevent hypothermia
Lab-Jack	Fisher scientific	14-673-50	4 x 4 in plate to adjust the height of the animal
X-Y gliding stage	Amscope	GT100	for positioning the animal under microscope