Name: a murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion
Uploaded: 2020-11-12T15:00:00.000+00:00
Duration: 5 min 20 s
Description: Watch this Scientific Journal Video about A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion at JoVE.com

This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (18K09424 to Toshihide Kurihara and 20K18393 to Yukihiro Miwa) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Keio University School of Medicine.
1. Preparation of surgical instruments and animals
<ol>
	<li>Autoclave surgical instruments and keep them in 70% ethyl alcohol. Prior to each new surgical procedure, clean surgical instruments carefully using 70% ethyl alcohol.</li>
	<li>Prepare male BALB/cAJc1 mice (6 weeks old, 26-28 kg) in a specific-pathogen-free (SPF) room to maintain sterile conditions before, during and after the surgery.</li>
</ol>
2. Transient bilateral common carotid artery occlusion (tBCCAO)
<ol>
	<li>Put a mouse under anesthesia via intraperitoneal injection with a combination of midazolam (40 &#956;g/100 &#956;L), medetomidine (7.5 &#956;g/100 &#956;L) and butorphanol tartrate (50 &#956;g/100 &#956;L), termed &#8220;MMB&#8221;, as previously described8,9. Hold the mouse&#8217;s back skins to keep the mouse away from bumping its eyes until the mouse is completely anesthetized.
	<ol>
		<li>Judge the depth of anesthesia by pinching the mouse toe until it has no response, of which method is commonly used for checking complete anesthesia10. 
		NOTE: Generally, less than 5 min are required for mice to fall asleep. Proper recipes for general anesthesia may be different by institutions.</li>
	</ol>
	</li>
	<li>Apply one drop of 0.1% purified sodium hyaluronate eye drop solution to the eyes to prevent dryness on the eyes under anesthesia.</li>
	<li>Place the mouse on its back and fix the mouse&#39;s paws using adhesive tapes.</li>
	<li>Disinfect the neck area of the mouse using 70% ethyl alcohol before the surgery. 
	NOTE: Additional clipping of the fur was not performed as this may cause subsequent skin inflammation11,12.</li>
	<li>Perform sagittal incision of the neck with a blade (Figure 1). 
	NOTE: Incision needs to be made on the midline between the neck, sternum and trachea.</li>
	<li>Separate both salivary glands carefully using two forceps and mobilize them to visualize the underlying CCAs.</li>
	<li>Isolate the right CCA carefully from the respective vagal nerves and accompanying veins without harming their structures, and place two 6-0 silk sutures under the CCA. Tie the two ties tightly to block the blood flow (Figure 1). 
	NOTE: During the procedure, small veins could be damaged. If bleeding is seen, wiping is required to visualize the CCAs clearly.</li>
	<li>Find the left CCA carefully from the respective vagal nerves and accompanying veins without harming their structures, and occlude the left CCA for 2 seconds by a clamp (Figure 1). 
	NOTE: A 6-0 silk suture needle is needed to be placed under the left CCA to mark a site for clamping.</li>
	<li>After reopening of the left CCA, suture wounds of the neck by a 6-0 silk suture and apply a dab of antibiotic (50 &#956;L) onto the neck to inhibit bacterial infection. 
	NOTE: Softly remove a clamp to avoid damaging the arterial wall when reopening of the left CCA.</li>
	<li>Inject 0.75 mg/kg of atipamezole hydrochloride intraperitoneally to the mouse to help the mouse recovered from deep anesthesia quickly. Return the mouse to a mouse cage with pre-heated pads. 
	NOTE: Do not let the mouse left unattended until the mouse regains sufficient consciousness to maintain sternal recumbency.</li>
	<li>Inject 0.4 mg/kg of butorphanol tartrate to the mouse for the management of pain when the mouse wakes up. 
	NOTE: The protocol can be paused here. As a first hint for successful tBCCAO, eyelid drooping of the mouse can be observed (Figure 2).</li>
	<li>For euthanasia, inject 3x of MMB mixture to the mice and sacrifice them for experiments.</li>
</ol>
3. General observations (survival rates and eyelid drooping)
<ol>
	<li>After the surgery, check survival rates for all causes of death at day 0 (after the surgery), 1, 3 and 7.</li>
	<li>Assess eyelid drooping by a 4-point rating scale: 1 = no drooping, 2 = mild drooping (~50%), 3 = severe drooping (over 50%), and 4 = severe drooping with eye discharge.</li>
</ol>
4. Retinal blood perfusion
<ol>
	<li>Inject 200 &#956;L of FITC-dextran (25 mg/mL) into the left ventricle of the mouse, which is commonly used for the observation of blood perfusion in mouse retinal vessels13,14.</li>
	<li>2 minutes after circulation, enucleate the eyes and fix in 4% paraformaldehyde for 1 hour. The retinas were carefully obtained and flat-mounted, as previously described15, and examined via a fluorescence microscope.</li>
	<li>Take photographs of the retinal whole mounts at 4x magnification and merge into a single using a merge analyzer, previously described16.</li>
	<li>Measure the perfused areas via a vessel analysis tool in NIH Fiji/ImageJ software.</li>
</ol>
5. Western blot
<ol>
	<li>3 and 6 hours after tBCCAO, obtain the eyes of mice and immediately transfer to a Petri dish containing cold PBS to isolate the retinas.</li>
	<li>After isolation of the retinas, perform western blotting, as previously described9.</li>
	<li>Incubate with antibodies for hypoxia-inducible factor-1&#945; (HIF-1&#945;; a general hypoxia marker) and for &#946;-Actin (an internal loading control) overnight followed by incubation of HRP-conjugated secondary antibodies. Visualize the signals via chemiluminescence.</li>
</ol>
6. Quantitative PCR (qPCR)
<ol>
	<li>6, 12 and 24 hours after tBCCAO, process the obtained retinas for qPCR, as previously described17.</li>
	<li>Perform qPCR via real-time PCR system. Primers used are listed in Table 1. Calculate fold changes between levels of different transcripts by the &#916;&#916;CT method.</li>
</ol>
7. Immunohistochemistry (IHC)
<ol>
	<li>3 days after tBCCAO, obtain the eyes of mice and embed in paraffin.</li>
	<li>Cut the paraffin-embedded eyes by a microtome to obtain the eye sections.</li>
	<li>De-paraffinize and stain the eye sections of 5 &#956;m thickness as previously described13.</li>
	<li>Incubate with an antibody for glial fibrillary acidic protein (GFAP; a reliable marker for astrocytes and M&#252;ller cells in the retina) overnight followed by incubation of Alexa Fluor 555-conjugated secondary antibody.</li>
	<li>Use DAPI (4&#8242;,6-diamidino-2-phenylindole) for staining the nucleus in the retina. Visualize signals via a fluorescence microscope.</li>
	<li>Assess morphology scoring by a 4-point rating scale, as previously described13,18: 0 = no signal, 1 = few positive glial end-feet in the ganglion cell layer (GCL), 2 = few labelled processes reaching from GCL to the outer nuclear layer (ONL), and 3 = most labelled processes reaching from GCL to ONL.</li>
</ol>
8. Electroretinography (ERG)
<ol>
	<li>3 and 7 days after tBCCAO, perform ERG using a Ganzfeld dome, acquisition system and LED stimulators, as previously described9.</li>
	<li>Following dark adaptation overnight, anesthetize mice with a combination of MMB under dim red light.</li>
	<li>Use a mixed solution of 0.5% tropicamide and 0.5% phenylephrine to dilate the pupils.</li>
	<li>Place the active electrodes on contact lens and place the reference electrode in the mouth.</li>
	<li>Obtain ERG responses from both eyes of each animal.</li>
	<li>Record scotopic responses under dark adaptation with various stimuli.</li>
	<li>Measure the amplitudes of a-wave from the baseline to the lowest point of a-wave.</li>
	<li>Measure the amplitudes of b-wave from the lowest point of a-wave to the peak of b-wave.</li>
	<li>Keep all mice warm during the procedure using heat pads.</li>
</ol>
9. Optical coherence tomography (OCT)
<ol>
	<li>2 weeks after tBCCAO, perform OCT using SD-OCT system, as previously reported8,9.</li>
	<li>For the measurement, subject mice to mydriasis by a mixed solution of 0.5% tropicamide and 0.5% phenylephrine, and to general anesthesia by a mixture of MMB.</li>
	<li>Obtain B scan images from equatorial slices of en-face scans.</li>
	<li>Examine the retinas at 0.2, 0.4 and 0.6 mm from the optic nerve head.</li>
	<li>Measure retinal thickness from the retinal nerve fiber layer (NFL) to the external limiting membrane (ELM), and consider the average of measured values as retinal thickness of an individual mouse.</li>
	<li>Plot the results as spider diagrams.</li>
</ol>

The authors have nothing to disclose.

In the study, we have shown that tBCCAO, using simple sutures and a clamp, could induce retinal ischemia and accompanying retinal dysfunction. Furthermore, we have demonstrated our current protocol for the development of a mouse model of retinal ischemia is easier and faster in comparison with other previous protocols for the development of retinal ischemic injury models2,3,7.
Anatomically, the left and right cerebral arteries can be connected via posterior communicating arteries (PCAs) which provide collateral circulation in the circle of Willis to maintain adequate blood supply to the central nervous system against flow interruption from occlusions or stenosis of individual vessels24,25 (Figure 1). Lee et al. demonstrated retinal blood perfusion could be 10 min-delayed (which is not an entire blockade of retinal blood perfusion in the ipsilateral retina) by permanent unilateral CCAO in C57BL6 mice13. This implies that induction of retinal ischemia by CCAO is closely associated with conditions of collateral circulation in the circle of Willis. C57BL6 is known to be the most susceptible mouse strain to cerebral ischemia by BCCAO among seven mouse strains including our current study&#8217;s mouse strain BALB26. Due to the incomplete circle of Willis in C57BL6, interruption of brain blood supply from both CCAs induces severe damages in the central nervous system, finally leading to death. In addition, in our preliminary study, we failed to induce tBCCAO in C57BL6 as almost all of the mice (about 80%) died within 3 days after the surgery (Supplementary Figure 4). Therefore, we applied tBCCAO to another mouse strain BALB for our current study.
To induce acute retinal ischemic injuries in our BALB model, the right CCA was permanently ligated and the left CCA was applied to boost acute retinal ischemic stress through transient occlusion. This is because mice could not tolerate ischemic stress induced by permanent BCCAO unlike rats who have the complete circle of Willis27. Next, we attempted to optimize occlusion time: left CCAO (0-20 seconds), as occlusion time has been considered one of key factors that impacts ischemic injuries to the central nervous system and directly connects with survival rates of experimental models28,29. We found that survival rates of BALB mice decreased in an occlusion time-dependent manner (Supplementary Figure 4). Occlusion of the left CCA over 10 seconds showed severely higher death rates (over 50%), while occlusion of the left CCA for 2 seconds or no occlusion (or unilateral CCAO) showed relatively higher survival rates (over 80%). Therefore, we excluded the groups (of occlusion time which is 10 and 20 seconds) for the further experiments as efficient and cost-effective experiments cannot be available. Next, we examined whether occlusion of the left CCA for 2 seconds or no occlusion (or unilateral CCAO) could induce retinal hypoxia. HIF-1&#945; is a major regulator functioning in hypoxic responses and is stabilized under hypoxic conditions30. In this regard, HIF-1&#945; stabilization has been used as a general molecular biological marker for hypoxia. We could not detect HIF-1&#945; stabilization in the retina in the group of unilateral CCAO (Supplementary Figure 5). Interestingly, we could detect HIF-1&#945; stabilization in the group of 2 seconds of tBCCAO (Figure 3). This implies retinal hypoxic stress could be induced by 2 seconds of tBCCAO in BALB mice. Therefore, 2 seconds of clamping time were finally selected for our study based on high survival rates after the surgery and induction of retinal ischemia via HIF-1&#945; stabilization.
Although the right CCA was permanently occluded in tBCCAO-operated mice, blood perfusion was partially detected in the right retina 2 minutes after systemic circulation of FITC-dextran (Supplementary Figure 1). Furthermore, we found that an alteration in HIF-1&#945; stabilization was not detected in the right retina in the unilateral CCAO-operated BALB mice. This phenomenon could be explained by effects of collateral circulation through the circle of Willis to maintain blood supply to the retina (Figure 1). Even though we could not clearly understand effects of left transient CCAO on blood perfusion to the right retina, transient left CCAO along with permanent right CCAO may boost acute hypoxic insults in the right retina as evidenced by a significant change in HIF-1&#945; expression in the right retina after tBCCAO (Figure 3). In addition, survival rates of mice were dependent on occlusion time of the left CCA. Taken together, the intensity of retinal ischemic stress could be controlled via left CCAO.
The phenotype of a droopy eyelid has been suggested as a presenting sign or a pathophysiological symptom of severe neurological conditions, especially ischemic stroke31,32. The muscle associated with a droopy eyelid is levator palpebrae superioris33. This muscle is supplied by the lateral palpebral artery which is one of branches derived from OpA. Hence, when OpA, which supplies the retina, is affected, eyelid drooping could be seen. Eyelid drooping was observed in MCAO mouse models34, which was also reproduced in our tBCCAO model. Moreover, we described that eyelid drooping becomes severe when occlusion time of the left CCA takes longer (Figure 2 and Supplementary Figure 6). This implies that the severity of eyelid drooping (indirectly referred to as the intensity of retinal ischemic stress) could be dependent on occlusion time of left CCA.
Retinal dysfunction is one of results seen in retinal ischemic retinopathies including stenosis of BCCA in mice35 and BCCAO in rats36. We found that the amplitudes of b-wave decreased in the tBCCAO-operated mice. Several previous studies demonstrated that MCAO also caused a reduction in the amplitude of b-wave after the surgery37,38. b-wave reflects a physiological condition of cells in retinal inner layers, including bipolar cells and M&#252;ller cells39. Furthermore, reactive gliosis by M&#252;ller cells was detected in the inner retinal layer after tBCCAO. This result is also reproduced in MCAO models40,41 and other CCAO models13,42. Taken together, it implies that inner retinal dysfunction could be induced by tBCCAO. Retinal thickness has been reported to increase transiently in acute retinal ischemia43,44. We also reproduced this finding in the tBCCAO-operated mice. This data shows that the impairment of blood circulation by tBCCAO could reach the retina and finally affect retinal layers.
For consistent outcomes, anesthetic time and length of surgical procedures as well as other factors such as weights and ages of experimental models and their body temperatures during and after the surgery should be standardized45. Particularly, attention is needed to maintain body temperature of mice throughout the experimental observation period. This is because hypothermia could have a preconditioning effect and interfere with ischemic effects by tBCCAO46. Even though we could not measure exact body temperature of the mice in our experiments, we used heating pads to warm the mice until the mice regained sufficient consciousness. Furthermore, we compared the tBCCAO-operated mice with the sham-operated mice to control potential confounding effects of uncontrollable factors.
Mouse strains could be an additional important variable factor to induce retinal ischemic injury by tBCCAO. Substantial variation of the circle of Willis in mouse strains could result in an unwanted reduction or induction of cerebral ischemia including retinal ishcemia47 and thereby could lead to variabilities of the results. Adjustment of clamping time is recommended for a successful tBCCAO-induced ischemic retinopathy when other strains of mice are required to be applied.
In general, incidents of stroke or other brain injuries are invariably accompanied with temporary or permanent vison loss48. To date, MCAO mouse model is widely used for stroke studies. As OpA originates proximal to the origin of MCA, any hindrance in the blood flow in MCA obstructs the flow to the retina. Retinal ischemia was firstly demonstrated in rats by MCAO37. Later, the same retinal ischemic model was applied to mice49. However, for the procedure, occlusion takes more than 60 minutes and finding an occlusion site is extremely difficult as MCA is buried deep inside the brain. Furthermore, the filament size and insertion length for MCAO greatly decides success of the surgery. Those additional variable factors induce variabilities of the ischemic outcomes after the surgery. Although direct comparison studies are needed between tBCCAO and MCAO, we described the beneficial features of our experimental models in this study: short occlusion time, simple experimental procedure and highly accessible occlusion sites. This model may solve the concerns seen in MCAO models.
While the use of the mouse model of retinal ischemia has great benefits for studying retinal ischemic injury, there remain limitations to this approach. Since surgical incision into the neck, separation of the salivary glands and occlusion in the right CCA with sutures must be applied for the procedure, the accompanying tissue disruptions can evoke associated inflammation systemically or at least locally. These concerns were partially addressed using the sham-operated mice, where all the surgical steps are all conducted without tBCCAO. Another issue is a requirement of managing pain that occurs during and after the surgery. In our study, pain management to prevent suffering of the mice was applied through the injection of butorphanol tartrate solution, a synthetically derived opioid agonist-antagonist analgesic of the phenanthrene series. It might be important to be aware that the use of different types of anesthetics and analgesics can disrupt effects of tBCCAO on retinal ischemia. Another limitation to this approach (along with the approaches of currently used other models) is that it does not provide a perfect simulation of pathologies associated with human cardiovascular retinal disorders. To date, mouse models used for such experiments do not suffer from co-morbidities that underlie ischemic retinopathies in humans, mainly with metabolic syndrome such as diabetes50. Such complications which are not present in current mouse models could have negative synergistic effects on the pathological pathways for the development of ischemic retinopathies. Therefore, this should be taken into account when interpreting the outcomes from the currently used experimental models including our tBCCAO mouse model. To better understand pathophysiological mechanisms of ischemic retinopathies in humans, our model can be combined with other pathological factors such as streptozotocin injection51 or high fat diet supplement52 for the development of ischemic diabetic retinopathy. At last, even though we showed reduced retinal blood perfusion in the tBCCAO-operated mice, we could not clearly understand effects of left transient CCAO on blood perfusion to the right retina. This matter could be addressed using laser-Doppler which is typically used to confirm that occlusion has taken place and ischemia has occurred in vivo in real time53,54. This technique could be utilized for better understanding of retinal ischemia in an individual tBCCAO-operated mouse, regarding the collateral circulation in the circle of Willis.
Despite these limitations, our tBCCAO method described here represents an effective approach to produce retinal ischemia in mice. Studying retinal changes by tBCCAO helps to unravel pathological mechanisms of ischemic retinopathies in humans. Furthermore, we hope that tBCCAO mouse model could be used for in vivo drug screening.

The retina is a neurosensory tissue for visual function. Since a substantial amount of oxygen is needed for visual function, the retina is known as one of the highest oxygen demanding tissues in the body1. The retina is susceptible to vascular diseases as oxygen is delivered through blood vessels. Various types of vascular diseases, such as diabetic retinopathy and retinal blood vessel (veins or arteries) occlusion, can induce retinal ischemia. To investigate pathological mechanisms of retinal ischemia, reproducible and clinically relevant experimental models of retinal ischemia are considered necessary. Middle cerebral artery occlusion (MCAO) by insertion of an intraluminal filament is the most generally utilized method for the development of in vivo rodent models of experimental cerebral ischemia2,3. Due to the proximity of the ophthalmic artery (OpA) to MCA, MCAO models are also used simultaneously to understand the pathophysiology of retinal ischemia4,5,6. To induce cerebral ischemia along with retinal ischemia, long filaments are typically inserted through incision of the common carotid artery (CCA) or the external carotid artery (ECA). These methods are difficult to perform, require a long time to complete the surgery (over 60 minutes for one mouse), and lead to high variabilities in the outcomes after the surgery7. It remains important to develop a better model to improve these concerns.
In this study, we simply used short transient bilateral CCA occlusion (tBCCAO) with needles and a clamp to induce retinal ischemia in mice and analyzed typical results of ischemic injuries in the retina. In this video, we will give a demonstration of the tBCCAO procedure.

<table><tbody><tr><td>Atipamezole hydrochloride</td><td>Zenoaq</td><td>Antisedan</td><td>For anti-anesthesia</td></tr><tr><td>Applied Biosystems 7500 Fast</td><td>Applied Biosystems</td><td>-</td><td>For qPCR</td></tr><tr><td>Butorphanol tartrate</td><td>Meiji Seika Pharma</td><td>Vetorphale</td><td>For anesthesia</td></tr><tr><td>BZ-II Analyzer</td><td>KEYENCE</td><td>-</td><td>For an image merge</td></tr><tr><td>BALB/cAJc1</td><td>CLEA</td><td>-</td><td>Mouse strain</td></tr><tr><td>&amp;beta;-Actin (8H10D10) Mouse mAb</td><td>CST</td><td>3700</td><td>For western blot</td></tr><tr><td>Clamp Forcep</td><td>World Precision Instruments</td><td>WPI 500451</td><td>For surgery</td></tr><tr><td>Dumont forceps #5</td><td>Fine Science Tools</td><td>11251-10</td><td>For surgery</td></tr><tr><td>DAPI solution</td><td>Dojindo</td><td>340-07971</td><td>For IHC</td></tr><tr><td>Envisu SD-OCT system</td><td>Leica</td><td>R4310</td><td>For OCT</td></tr><tr><td>FITC-dextran</td><td>Merk</td><td>FD2000S</td><td>For retinal blood perfusion</td></tr><tr><td>Fluorescence microscope</td><td>KEYENCE</td><td>BZ-9000</td><td>For fluorescence detection</td></tr><tr><td>Gatifloxacin hydrate</td><td>Senju Pharmaceutical</td><td>Gachifuro</td><td>For anti-bacterial infection</td></tr><tr><td>GFAP Monoclonal Antibody (2.2B10)</td><td>Thermo</td><td>13-0300</td><td>For IHC</td></tr><tr><td>Heating pad</td><td>Marukan</td><td>RH-200</td><td>For surgery</td></tr><tr><td>HIF-1&amp;alpha; (D1S7W) XP Rabbit mAb</td><td>CST</td><td>36169</td><td>For western blot</td></tr><tr><td>ImageQuant LAS 4000 mini</td><td>GE Healthcare</td><td>-</td><td>For chemiluminescence</td></tr><tr><td>Midazolam</td><td>Sandoz K.K</td><td>SANDOZ</td><td>For anesthesia</td></tr><tr><td>Microtome Tissue-Tek TEC 6</td><td>Sakura</td><td>-</td><td>For sectioning</td></tr><tr><td>Medetomidine</td><td>Orion Corporation</td><td>Domitor</td><td>For anesthesia</td></tr><tr><td>Needle holder</td><td>Handaya</td><td>HS-2307</td><td>For surgery</td></tr><tr><td>PuREC</td><td>MAYO Corporation</td><td>-</td><td>For ERG</td></tr><tr><td>Scissor</td><td>Fine Science Tools</td><td>91460-11</td><td>For surgery</td></tr><tr><td>Sodium hyaluronate</td><td>Santen Pharmaceutical</td><td>Hyalein</td><td>For eye lubrication</td></tr><tr><td>Tropicamide/Penylephrine hydrochloride</td><td>Santen Pharmaceutical</td><td>Mydrin-P</td><td>For mydriasis</td></tr><tr><td>6-0 silk suture</td><td>Natsume</td><td>E12-60N2</td><td>For surgery</td></tr></tbody></table>

a murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

After systemic circulation of FITC-dextran for 2 minutes, retinal vasculatures of the left and right retinas in the sham-operated mice and tBCCAO-operated mice were examined (Supplementary Figure 1). FITC-dextran was fully visible in the both retinas in the sham-operated mice and the left retina in the tBCCAO-operated mice, while it was partially detectible in the right retina in the tBCCAO-operated mice.
After tBCCAO, eyelid drooping was examined (Figure 2). The right eyes showed mild (score 2; 75%) and severe eyelid (score 3 and 4; 25%) drooping, while the left eyes had no drooping (score 1; 93.75%) except for one mouse (score 2; 6.25%). Although severe eyelid drooping with eye discharge was not considerably observed in the tBCCAO-operated mice, we could see one mouse for this phenotype (score 4; 6.25%).
Reduced oxygen status in tissues leads to stabilization of HIF-1&#945; and induction of a number of hypoxia-responsive genes such as EPO, VEGF and BNIP319,20,21. First of all, molecular biological hypoxia using a general hypoxic marker HIF-1&#945; was evaluated via western blotting (Figure 3). Increased HIF-1&#945; expression was significantly observed in the right retina 3 and 6 hours after tBCCAO. Next, expressions of hypoxia-responsive genes were evaluated via qPCR (Supplementary Figure 2). There was no significant change in hypoxia-responsive gene expressions 6 hours after tBCCAO. 12 hours after tBCCAO, we found Binp3 expression significantly increased and a slight increase in Epo expression was shown in the right retina. 24 hours after tBCCAO, we could also find a slight increase in Epo expression in the right retina although it was not statistically significant. Vegf expression was not altered from 6 to 24 hours in the tBCCAO-operated mice.
Retinal reactive gliosis was examined 3 days after tBCCAO (Figure 4), as glia such as astrocytes and M&#252;ller cells have been closely associated with retinal ischemia22. GFAP has been widely used for detection of astrocytes and M&#252;ller cells in the retina23. The average of morphology scores for GFAP labelling in the right retina was the highest among the both retinas in the sham-operated mice and the left retina in the tBCCAO-operated mice. Based on the localization of GFAP expression, a change in morphology in GFAP labelling is considered to reflect activation of M&#252;ller cells.
ERG was used to examine retinal dysfunction after tBCCAO (Figure 5). The amplitudes of b-wave in the right eye dramatically decreased 3 and 7 days after tBCCAO. However, the amplitudes of a-wave in the right eye were not significantly changed. When it comes to the left eye, we could not see any changes in the amplitudes of a- and b-waves (Supplementary Figure 3).
We performed OCT to determine an alteration in retinal thickness after tBCCAO (Figure 6). Retinal thickness in the right eye dramatically increased 2 weeks after tBCCAO, while there was no difference in retinal thickness in the left eye between the tBCCAO- and sham-operated mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61865/61865fig01.jpg" /> 
Figure 1: Schematic of the model procedure and blood circulation in the circle of Willis.&#160;A schematic illustration showed the tBCCAO-induced retinal ischemic mouse model procedure and blood circulation to the retina. CCA, ECA, ICA, PCA and OpA represent the common carotid artery, external carotid artery, internal carotid artery, posterior communicating artery and ophthalmic artery, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/61865fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61865/61865fig02.jpg" /> 
Figure 2: Eyelid drooping after tBCCAO.&#160;The severity of eyelid drooping was assessed by 4-point rating based on the reference images: 1 = no drooping, 2 = mild drooping (~50% drooping), 3 = severe drooping (over 50% drooping), and 4 = severe drooping with eye discharge. Eyelid drooping was observed after tBCCAO and it was maintained during the experimental observation. The results (sham: n = 10, tBCCAO: n = 16) were plotted as a scatter dot plot. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/61865fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61865/61865fig03.jpg" /> 
Figure 3: HIF-1&#945; stabilization after tBCCAO.&#160;Representative immunoblots and quantitative analyses (groups for hour 3; sham: n = 3, tBCCAO: n = 6 and groups for hour 6; sham and tBCCAO: n = 6) for HIF-1&#945; and &#946;-Actin showed that HIF-1&#945; was stabilized in the right retina 3 and 6 hours after tBCCAO. *P &#60; 0.05. The data were analyzed using Student&#8217;s t-test and presented as mean with &#177;standard deviation. L and R stand for the left and right retina, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/61865fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61865/61865fig04.jpg" /> 
Figure 4: Reactive gliosis after tBCCAO.&#160;Representative sagittal sections of the retinas (sham: n = 4, tBCCAO: n = 4) and quantitative analyses of GFAP labelling (red) by a morphology scoring (0&#8211;3) showed that GFAP labelling, mostly restricted in NFL+GCL, was expanded to the entire inner layer, from GCL to ONL (white arrows) in the right retina after tBCCAO. Scale bars, 50 &#956;m. DAPI (blue) was used for staining the nucleus in the retina. NFL, GCL, IPL, INL and ONL represent the nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer and outer nuclear layer, respectively. The data was analyzed using Student&#8217;s t-test and presented as median with interquartile range, the 25th and 75th percentile. *P &#60; 0.05. L and R stand for the left and right retina, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/61865fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61865/61865fig05.jpg" /> 
Figure 5: Visual dysfunction in the right eye after tBCCAO.&#160;(A) Representative waveforms of dark-adapted ERG performed 3 and 7 days after tBCCAO. Stimulation intensity (cd.s/m2): 0.005. (B) Quantitative analyses showed that there was a decrease in the amplitudes of b-wave in the right eye (sham: n = 5, tBCCAO: n = 6) while the amplitudes of a-wave were not changed. *P &#60; 0.05, **P &#60; 0.01. The data were analyzed using Student&#8217;s t-test and presented as mean with &#177;standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/61865fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61865/61865fig06.jpg" /> 
Figure 6: A change in retinal thickness after tBCCAO.&#160;Representative OCT images in the sham- and tBCCAO-operated retinas and quantitative analyses showed that there was an increase in retinal thickness in the right retina (sham: n = 4, tBCCAO: n = 8). There was no change in retinal thickness in the left retina (sham: n = 4, tBCCAO: n = 8). Scale bars are 200 (upper) and 100 (lower) &#181;m, respectively. *P &#60; 0.05. The values in the horizontal axis of the diagrams represent 0.2, 0.4 and 0.6 mm distant from the optic nerve head (0) which was detected by the green line. The data were analyzed using two-way ANOVA followed by a Bonferroni post hoc test. Spider diagrams were presented as mean with &#177; standard deviation. NFL, INL, ONL and ELM are the nerve fiber layer, inner nuclear layer, outer nuclear layer and external limiting membrane, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/61865fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Retinal blood perfusion after tBCCAO. Representative retinal flat mount images (with higher-magnification of each image) after 2 min of FITC-dextran circulation and quantitative analyses showed that full perfusion was observable in the both retinas in the sham-operated mice and the left retina in the tBCCAO-operated mice. However, the right retina in the tBCCAO-operated mice showed partial blood perfusion. The data were analyzed using Student&#8217;s t-test and presented as mean with &#177;standard deviation. L and R stand for the left and right retina, respectively. Scale bars are 800 and 400 &#956;m, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: Expressions of hypoxia-responsive genes after tBCCAO. Quantitative analyses showed a transient increase in Bnip3 mRNA expression in the right retina with statistical significance 12 hours after tBCCAO. Epo mRNA expression showed an increasing tendency in the right retina for 24 hours after tBCCAO, although its values were not significantly different in comparison with the sham-operated right retina. **P &#60; 0.01. The data were analyzed using Student&#8217;s t-test and presented as mean with &#177;standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 3: Visual function in the left eye after tBCCAO. Quantitative analyses showed that there was no change in the amplitudes of a- and b-waves in the left eye (sham: n = 5, tBCCAO: n = 6). P &#62; 0.05. The data were analyzed using Student&#8217;s t-test and presented as mean with &#177;standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/supfig03.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 4: Survival rates after tBCCAO in C57BL6 and BALB. Kaplan-Meier survival curves demonstrated that almost all mice died within 3 days after tBCCAO in C57BL6 mice. When it comes to BALB mice, longer clamping time in tBCCAO induces sudden and severe animal death (survival rates on day 7, 20 sec: 10%, 10 sec: 20%, 2 sec: 81%, and 0 sec: 95%). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/supfig04.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 5: HIF-1&#945; stabilization after unilateral CCAO. A representative immunoblot and quantitative analysis (sham: n = 3, unilateral CCAO: n = 3) for HIF-1&#945; and &#946;-Actin showed that HIF-1&#945; was not stabilized in the retinas 3 hours after unilateral CCAO. P &#62; 0.05. The data was analyzed using Student&#8217;s t-test and presented as mean with &#177;standard deviation. L and R stand for the left and right retina, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/supfig05.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 6: Severe eyelid drooping after tBCCAO with long clamping time. 10 seconds of tBCCAO induced severe eyelid drooping, which was assessed by a 4-point rating scale: 1 = no drooping, 2 = mild drooping (~50%), 3 = severe drooping (over 50%), and 4 = severe drooping with eye discharge, as described in Figure 2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61865/supfig06.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion at JoVE.com

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

a-murine-model-ischemic-retinal-injury-induced-transient-bilateral

Nanyang Technological University

Research

JoVE Journal

Medicine

7.4K Views.  Keio University School of Medicine. Hier beschrijven we een muismodel van retinale ischemie door voorbijgaande bilaterale gemeenschappelijke halsslagader occlusie met behulp van eenvoudige hechtingen en een klem. Dit model kan nuttig zijn voor het begrijpen van de pathologische mechanismen van retinale ischemie veroorzaakt door cardiovasculaire afwijkingen.

Video: A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

Neuroscience

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

Mouse Model for Retinal Ischemia-Reperfusion Injury to Study Retinal Pathologies

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (&#8804;20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

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