Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.
Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.
Since the 1980s, the middle cerebral artery occlusion (MCAO) model induced by silicone filaments has been widely used in basic stroke research1. Other methods (i.e., suturing of one branch of the MCA2 and photochemically induced focal infarction) have also been used. These models have been termed MCA-based stroke models and have greatly contributed to investigations of the pathophysiological mechanisms underlying stroke and potential therapeutics. Although there are limitations of these experimental models3,4, these methods have been used many labs5,6. MCA-based stroke models represent a stroke in the anterior circulation; however, few reports have investigated models mimicking stroke in the posterior circulation7.
There are significant differences among the etiology, mechanisms, clinical manifestation, and prognosis between anterior and posterior circulation strokes8. Therefore, the results derived from anterior circulation stroke models cannot be applied to posterior circulation stroke. For example, the reperfusion time window for anterior circulation has been extended to 6 h, with a small portion of studies extending to 24 h based on imaging findings9. However, the time window for posterior circulation may be longer than 24 h, according to previous reports10 and our own clinical experiences. This elongated reperfusion time window must be further studied and confirmed in experimental models.
Regarding posterior circulation strokes, pontine infarction is the most common subtype, accounting for 7% of all ischemic stroke cases11,12. According to infarction topography, pontine infarctions are divided into isolated and non-isolated pontine infarctions13. Isolated pontine infarctions are categorized into three types based on the underlying mechanisms: large artery disease (LAD), basilar artery branch disease (BABD), and small artery disease (SAD). Knowledge of the mechanisms, manifestation, and prognosis of pontine infarction has been derived from clinical investigations of cases14. However, a rodent model mimicking pontine infarction has been less investigated.
In previous studies, diffuse brainstem tegmentum injury involving the pons has been explored7. One group attempted to create a pontine infarction model via ligation of the basilar artery (BA)15. Another group used a 10-0 nylon monofilament suture to selectively ligate four points of the proximal BA selectively16. This model mimics LAD, but most pontine infarctions result from BABD and SAD. In addition, selective ligation of the BA is a complicated surgery and has a high death rate.
Provided here is a detailed protocol for an easy-to-perform, easily reproduced, and successful rat model of acute pontine infarction by electrical stimulation.
The protocol was reviewed and approved by the Institution Animal Care and Use Committee of The Second Affiliated Hospital of Guangzhou Medical University, an institution accredited by AAALACi. The rats were provided by the Animal Center of Southern Medical University.
1. Animal
2. Establishment of infarction in the pons
3. Behavioral tests
4. Infarct confirmation by MRI
5. Infarct confirmation by TTC staining
6. Statistics
Six animals were subjected to the surgery protocol described above. The control group as shown in the Figure 4 consisted of six rats. The brain slices shown in the Figure 4 were derived from one rat per group.
The MRI scanning showed that the infarction was located in the basis of the pons (Figure 4A). Since the probe was injected 2 mm to the left of the midline, the infarction was located laterally. This infarction mimics anterolateral pontine infarctions in patients (Figure 4A). Because an insulated sheath was used, there was no infarction beyond the tip of the probe including the cortex, cerebellum, and midbrain (Figure 4A). DWI images also revealed the acute pontine infarction (Figure 4A).
TTC staining was used to confirm the infarction 24 h post-surgery (Figure 4A). Compared to the control group, the infarction volume was significantly higher (Figure 4B).
Behavioral scores were measured before and after surgery. The scores for the control and infract model groups before and after surgery are presented in Table 1. Due to the lack of a specific behavioral test designed for pontine infarction, the Longa score, Berderson score, and balance beam test were used to assess the neurological deficits. Additionally, the adhesive removal somatosensory test to assess the sensorimotor function as well as limb-placement test to assess the proprioception.
Compared to the control group, the rats with pontine infarction circled to the left (Figure 4A). There were significant differences in Longa score (2.67 ± 0.52 vs. 0, p < 0.05, Figure 4C), Berderson score (2.67 ± 0.52 vs. 0, p < 0.05, Figure 4D), limb placement test (4.67 ± 0.52 vs. 0, p < 0.05, Figure 4E), beam balance test score (118.33 ± 2.66 vs. 10.17 ± 1.47, p < 0.05, Figure 4F), and adhesive removal somatosensory test score (2.33 ± 0.52 vs. 12.0 ± 0, p < 0.05, Figure 4G) between rats with pontine infarction and control group rats.
Figure 1: Infarction establishment. (A) A hole made in the skull. (B) The sheath is moved to the hole. (C) Injection of the sheath. (D) Injection of the probe. (E) The anode (red arrow) is connected. (F) The probe is removed. (G) Hole (red arrow) left in the brain surface. Please click here to view this video. (Right-click to download.)
Figure 2: Location of probe. (A) Schematic diagram of stereotaxic locations: arrows point to retraction of skin flaps, site of Bregma, and positioning of drill. (B) Schematic diagram of the sheath and probe. (C) Location of tip of sheath placed in the pons. (D) Location of tip of the probe placed in the pons. (E) Experimental design. Please click here to view this video. (Right-click to download.)
Figure 3: Lesion-producing device. (A) Separate of sheath and probe. (B) The probe in the sheath. (C) The blue electrode was anode which was connected to the caudal probe; the red electrode was cathode. (D) Electrical stimulator. (E) Surgical instruments. Please click here to view this video. (Right-click to download.)
Figure 4: Representative results. (A) The infarction was assessed by MRI scanning with T2 and DWI sequence in vivo and was confirmed by TTC staining in vitro 24 h after surgery. Acute pontine infarction located in the right anterolateral pons (dotted line). Behavioral test showed that the rat circled to the contralateral side of lesion. (B) The volume of infarction. (C) Long score. (D) Bederson score. (E) Limb placing test. (F) Balance beam walking test. (G) Adhesive removal somatosensory test. Bars represent mean ± SD (p < 0.05 vs. control group). Please click here to view this video. (Right-click to download.)
Figure S1: Lacunar infarction in the pons. The length of the probe tip is shortening. MRI scanning shows a lacunar infarction in the right pons. (A) T2 image. (B) DWI image. Please click here to view this video. (Right-click to download.)
Rat NO | Longa score | Berderson score | Balance beam test | Adhesive-removal somatosensory test | Limb-placement test | |||||
Pre | Post-surgery | Pre | Post-surgery | Pre | Post-surgery | Pre | Post-surgery | Pre | Post-surgery | |
Pontine infarction 1 | 0 | 3 | 0 | 2 | 0 | 5 | 6 | 120 | 12 | 2 |
Pontine infarction 2 | 0 | 2 | 0 | 3 | 0 | 4 | 8 | 120 | 12 | 3 |
Pontine infarction 3 | 0 | 3 | 0 | 3 | 0 | 5 | 8 | 116 | 12 | 2 |
Pontine infarction 4 | 0 | 3 | 0 | 3 | 0 | 4 | 6 | 120 | 12 | 2 |
Pontine infarction 5 | 0 | 3 | 0 | 2 | 0 | 5 | 7 | 114 | 12 | 2 |
Pontine infarction 6 | 0 | 2 | 0 | 3 | 0 | 5 | 7 | 120 | 12 | 3 |
Control 1 | 0 | 0 | 0 | 0 | 0 | 0 | 9 | 11 | 12 | 12 |
Control 2 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 10 | 12 | 12 |
Control 3 | 0 | 0 | 0 | 0 | 0 | 0 | 10 | 8 | 12 | 12 |
Control 4 | 0 | 0 | 0 | 0 | 0 | 0 | 7 | 11 | 12 | 12 |
Control 5 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 9 | 12 | 12 |
Control 6 | 0 | 0 | 0 | 0 | 0 | 0 | 9 | 12 | 12 | 12 |
Table 1: Behavioral scores.
The present study provides a protocol for generating an acute pontine infarction rat model. This model can be used for research on prognosis and rehabilitation (including post-stroke chronic pain) in pontine stroke patients.
There are several strengths of this method. First, it provides a rat model of acute pontine infarction for future studies. As mentioned above, pontine infarction is a common stroke subtype that has received less attention. A major shortcoming of stroke research has been the lack of a specific pontine infarction model. Second, in comparison to the existing pontine infarction rat model by ligation of the BA15,16, this model can be adjusted to alter the location and volume of the infarction according to the experimental design. For example, the length of the tip can be changed so that the infarction extends from the surface of the pons, as done here.
Alternatively, a lacunar infarction in the pons may be established by shortening the length of the probe tip (Supplemental Figure 1). Infarctions in different locations of the pons (i.e., anteromedial pontine infarction) and in different planes of the pons (i.e., upper, middle and lower planes) may also be created according to the topographic design. In this model, the upper pontine plane was chosen. Third, this model is easy to establish and possesses a high success rate. Ligation of the BA may not produce infarction due to the potential collateral circulation15, but this model establishes the infarction at a high success rate, which is essential for reliable research models.
There are some limitations of this method. First, the infarction in this model is not a real stroke. Stroke is a result of vascular vessel lesions, disturbance of blood contents, or dysfunction of regulation of cerebral blood flow. The infarction is created by a lesion in the pons that does not spontaneously occur. In other words, this model cannot be used to address why the stroke occurs in the pons. Second, this model requires special equipment, such as the lesion-producing device and stereotaxic apparatus.
In conclusion, the findings prove this model’s success in establishing an experimental acute pons stroke model. Based on this novel model, the resulting cell loss and prognosis of acute pontine infarction can be further investigated and allow for future therapeutic developments.
The authors have nothing to disclose.
This study was financially supported by the National Science Foundation of China (81471181 and 81870933) to Y. Jiang and the National Science Foundation of China (No. 81601011), Natural Science Foundation of Jiangsu Province (No. BK20160345) to J. Zhu and by the Scientific Program of Guangzhou Municipal Health Commission (20191A011083) to Z. Qiu.
4-0 sucture | Shanghai Jinzhong | Surgical instruments | |
Adhesive tape | Shanghai Jinzhong | Surgical instruments | |
Animal anesthesia system | RWD | Wear mask when using the system | |
Bone cement | Shanghai Jinzhong | Surgical instrument | |
Cured clamp | Shanghai Jinzhong | Surgical instrument | |
General tissue scissors | Shanghai Jinzhong | Surgical instrument | |
IndoPhors | Guoyao of China | Sterilization | |
Isoflurane | RWD | 217181101 | |
Lesion Making Device | Shanghai Yuyan | Making a lesion | |
MRI system | Bruker Biospin | Confirmation of infarction in vivo | |
Needle holder | Shanghai Jinzhong | Surgical instrument | |
Penicilin | Guoyao of China | Infection Prevention | |
Probe | Anke | Need some modification | |
Q-tips | Shanghai Jinzhong | Surgical instrument | |
Shearing scissors | Shanghai Jinzhong | Surgical instrument | |
Stereotaxic apparatus | RWD | ||
Suture needle | Shanghai Jinzhong | Surgical instrument | |
Tissue holding forcepts | Shanghai Jinzhong | Surgical instrument | |
TTC | Sigma-Aldrich | BCBW5177 | For infarction confirmation in vitro |