Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Podsumowanie

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Streszczenie

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Wprowadzenie

At the beginning of the 21th century, ischemic stroke is a devastating disorder that represents the second cause of long term disability¹ and the second cause of mortality worldwide, in which stroke accounted for approximately 5,7 million deaths in 2004². In spite of the many efforts that were put in, there is still no effective treatment available to improve functional recovery after stroke. Animal models of stroke are widely used in the field of stroke research as they allow modeling of the pathophysiology of ischemic damage and test the efficacy of different neuroprotective strategies in vivo. Most of these models aim to induce extensive infarctions by interrupting (temporarily or permanently) the blood flow within the middle cerebral artery, whereas other models were developed to study lesions of small size in specific areas, typically the motor and somatosensory cortices. However, several factors may contribute to generate a certain degree of variability in experimental stroke studies, including the mouse strain used, the age and sex of animals included in the study and, above all, the technique adopted to induce the ischemic damage. With regard to the latter point, the duration and invasiveness of the surgery (i.e. the need for a craniotomy) as well as the surgical skill required to the operator to reliably induce an ischemic lesion are critical determinants for a successful and unbiased in vivo stroke study.

The concept of photothrombosis was initially proposed by Rosenblum and El-Sabban in 1977³ and became renowned by its application in rat brain by Watson et al. in 1985⁴ in which the technique was largely improved and set the basis of the current model^3-6. The photothrombotic approach aims to induce a cortical infarction through the photo-activation of a light-sensitive dye previously delivered into blood system, which results in local vessel thrombosis in the areas exposed to the light. When the circulating dye is illuminated at the appropriate wavelength by a cold light source, it releases energy to oxygen molecules, which in turn generate a large amount of highly reactive singlet oxygen products. These oxygen intermediates induce endothelial cell membrane peroxidation, leading to platelet adhesion and aggregation, and eventually to the formation of thrombi which determine local cerebral flow interruption⁷.

Photothrombosis is a non-canonical ischemic model that does not occlude or break only one artery as it usually happens in human stroke, but induces lesions in more superficial vessels, which results in selective interruption of blood flow in the areas exposed to light. For this reason, this approach may be suitable for cellular and molecular studies of cortical plasticity. The principal advantage of this technique resides in its simplicity of execution. Moreover, photothrombosis can be easily performed in approximately forty minutes per animal, including twenty-minute wait (3 min for anesthesia; 1 min to shave the scalp; 3 to 5 min to place the animal on the stereotaxic apparatus; 2 min to scrub the scalp with antiseptic solution, make an incision and clean the skull; 2 to 4 min to place the cold light fiber; 1 min to inject the rose Bengal solution; 5 min-wait for intraperitoneal diffusion; 15 min of illumination; and 5 min to clean the wound and suture the animal). Furthermore, no surgical expertise is needed to perform this technique as the lesion is induced through simple illumination of the intact skull. Unlike classical arterial occlusion, this method determines selective occlusions of pial and intraparenchymal microvessels within the irradiated zone and reduces the variability among lesions as no collateral vessel is left to supply oxygen in the targeted area.

In spite of its particular nature, the photothrombotic damage shares essential mechanisms occurring in brain stroke. Similarly to artery occlusion in human stroke, platelet aggregation and clot formation determine interruption of blood flow in the irradiated area⁷. Likewise, this model also shares essential inflammatory responses as in middle cerebral artery occlusion⁸. However, because of the well-delimitated boundaries, the penumbral zone, which corresponds to an area of partially preserved metabolism, is very reduced or inexistent after a photothrombotic lesion. This clear border can facilitate the study of cellular responses within ischemic or intact cortical area. Photothrombosis mouse model is particularly suitable for stroke studies in a variety of transgenic animals. Indeed classical models cannot fit to all strains and long period studies in C57BL/6 mouse strain reported a high mortality ratio that can cause bias⁹.

Protokół

1. Pre-surgery

1. Weigh Rose Bengal in an 1.5 ml tube and dissolve in sterile saline solution until reaching a final concentration of 15 mg/ml. Filter sterilize through a 0.2 μm filter and store it in the dark at room temperature up to two months.
2. Sterilize all surgical instruments by autoclaving. The surgical area should be sanitized less than one hour before initiating the surgery.
3. Record the mouse body weight to adjust the dose of Rose Bengal to be injected. We injected 10 μl/g animal weight in female CD1 mice 12 weeks old in the present protocol. The amount of Rose Bengal needed to produce the desired cortical lesion size can be easily determined in a separate set of preliminary experiments by testing different dosages (typically 2 μl/g, 5 μl/g or 10 μl/g body weight). Note that the amount of Rose Bengal to be injected is highly dependent on the experiment conditions, particularly the type of light source used and the duration of light exposure. In the present study, 10 μl/g (150 μg/g) dose was found to be necessary to induce photothrombosis upon exposure to light for 15 min, whereas others groups reported that either 50 μg/g ^6,8 and 100 μg/g¹⁰ intraperitoneal injection was sufficient for inducing a photothrombotic lesion.

2. Anesthesia Procedure

1. Anesthetize mice with isoflurane in a transparent induction chamber (3.5-4% for induction, 1.5-2% for maintaining) in 50% (v/v) oxygen/50% (v/v) dinitrogen monoxide gas mixture. Gaseous anesthesia allows a quick wake up of the animals and the level of anesthetic gas can be adjusted easily. Alternatively, mice can also be anesthetized by a ketamine-xylazine mixture.
2. When deep anaesthesia is reached, remove the anesthetized animal from the induction chamber, place it in the stereotaxic frame and maintain anesthesia using a face mask. Adjust the isoflurane dose to achieve an adequate anesthesia level. Monitor the breath rate throughout the procedure and make sure it is constant (40- 60 breaths per minute).
3. Use toe pinches in order to ensure the animal is deeply anaesthetized.
4. Apply eye ointment, in order to prevent the eyes from drying out.
5. Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad for maintaining the mouse body temperature at 37 ± 0.5 °C.

3. Surgery for Illumination of the Target Area

1. Shave the mouse scalp with an electric razor.
2. Firmly secure the head and insert the ear bars into external meatus. Be careful not to damage eardrums.
3. Disinfect the skin on the surface with alternating swipes of 70% ethanol and betadine using cotton swabs.
4. Use a scalpel to make an incision along the midline from the eye level down to the neck. Apply skin retractors to keep the skull exposed.
5. Gently retract the periostium to the edges of the skull with a scalpel and let the skull surface dry using sterile cotton swabs. Identify the bregma and lambda. Place a glass micropipette on the bregma as reference point, then move it to the coordinates of your region of interest. The region of interest chosen for this article is roughly roundly shaped, centered approximately 2 mm lateral to the bregma, and covering an area of about 30 mm^2, which includes a large part of the sensorimotor cortex according to the mouse brain atlas by Franklin and Paxinos¹¹.
6. Mark the position of interest with reference points and put an optic fiber in close contact with the skull surface to avoid light scattering but pay attention not to exert pressure on it. The illuminated area can be restricted by applying on the skull a mask with small aperture or a cap at the tip of the optic fiber guide.

4. Rose Bengal Injection and Activation

1. Load the Rose Bengal solution in a 1 ml syringe and calculate the amount to be injected according to a dose of 10 μl/g of body weight.
2. Proceed to a slow intraperitoneal injection.
3. Let the dye diffuse and enter the blood stream. After 5 min, switch on the cold light illuminator. Avoid illumination of the animal by any other source of light. We used fiber optic illuminator of 150 W intensity.
4. After 15 min of illumination, stop light exposure and suture the wound. Five minutes of illumination already produce infarction and 10 min is likely sufficient to obtain a maximal effect¹². In order to minimize variability, we choose to illuminate for 15 min.

5. Suture

1. Remove the skin retractors and apply sterile saline solution to avoid dehydration.
2. Close up the wound using reverse cutting needle and silk or nylon suture thread.
3. Interrupt anesthesia delivery, carefully remove the mouse from the stereotaxic apparatus and put it on a pre-warmed heating pad until it is fully awake, then return it to its cage. Body temperature should be monitored carefully throughout the procedures to limit the variability in the infarct extension. According to the concentration and the route of administration, Rose Bengal can still be detected in the blood stream for several hours after injection¹³. Prefer the heating blanket to warming lamp to avoid potential secondary damages (Rose Bengal absorption wavelength is in the green spectrum).

Wyniki

This protocol will produce a cortical lesion that is already visible upon dissection of the cortex to the unaided eye (Figures 1A-1C). The photothrombotic lesion develops in superficial and deep cortical layers in which the tissue is sufficiently translucent to allow photo-activation of the Rose Bengal. Measurement of the extent of cerebral infarction can be performed quickly by histological staining with triphenyl-tetrazolium chloride (TTC) on fresh tissue or by cresyl violet after fixation in 4% parafo...

Dyskusje

Modifications and substitutions

Because of its absorption peak at 562 nm, a green light laser from a filtered xenon arc lamp was originally chosen to irradiate the photosensitive Rose Bengal. Although laser-mediated excitation was still used recently5, it can be replaced by cold light lamp that also ensure dye excitation^10,15. Cold light optic fibers are easier to manipulate and less expensive than laser sources. However, it should be noticed that lasers are commonly used to target ind...

Materiały

Name	Company	Catalog Number	Comments
Solutions and chemicals
Rose Bengal	Sigma, Italy	330000
Isoflurane Vet	Merial	103120022
Betadine	Asta Medica
Paraformaldehyde	Sigma-Aldrich	158127
Surgical material and equipment
Fluosorber Filter	Havard apparatus	340415
150W fiber optic illuminator	Photonic	PL3000
Temperature Controller for Plate TCAT-2DF	Havard apparatus	727561
Stereotaxic Instrument	Stoelting	51950
Operating microscope	Takagi	OM8
Heating pad
Oxygen and nitrogen gas
Surgery Tools	World precision instrument		Optic fiber taps and mask are custom-made