We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.
We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 µL of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.
Intracerebral hemorrhage accounts for one-fifth of stroke patients. The prognosis of intracerebral hemorrhage depends on the speed, volume, and location of bleeding1,2. Compared to the forebrain hemorrhage, the brainstem hemorrhage has higher mortality and morbidity3. About 40% of brainstem hemorrhage occurs in the pons4. The etiology and pathophysiology of pontine hemorrhage are quite different and less studied than forebrain hemorrhage5.
There are two kinds of pontine hemorrhage animal models. One is spontaneous hemorrhage model induced by infusion of bacterial collagenase in the pons6,7,8. The biggest advantage of this model is that the bleeding is spontaneous. However, collagenase can only induce a small volume of pontine hemorrhage. Besides, collagenase might cause other injuries to the brain. The other model is induced by stereotactic injection of autologous blood into the pons9. The advantage of this model is that it is easy to master with a high success rate. Theoretically, researchers could inject any volume of blood into any location of pons. However, due to the back-leakage through the needle route, the injected volume is limited. Recently, the double-injection method has been promoted to reduce the back-leakage9. This method injects autologous blood twice with a 20-minute interval between the injections. The double-injection method is applied to induce mild (30 µL) and moderate (60 µL) pontine hemorrhage but not massive pontine hemorrhage. In the clinic, the majority of pontine hemorrhage patients with poor prognosis have massive hemorrhage (more than 10 mL).
In the previous study, we provided a protocol to establish a pontine ischemic stroke model in rat10. In this study, we modify the existing dual injection method, and provide a detailed protocol to induce massive pontine hemorrhage in a rat by dual injection of 100 µL autologous blood at two different locations in the pons.
The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Guangzhou Medical University. Rats were provided by the Animal Center of Southern Medical University. The experimental design is shown in Figure 1.
1. Animal and instruments
2. Inject the blood in the pons
3. Behavioral tests
NOTE: Perform behavioral tests on Day 1, Day 3, Day 7, and Day 14 after modeling, including the balance beam test, limb placement test, and the modified Voetsch neuroscore.
4. Hemorrhage confirmation by MRI
5. Hemorrhage confirmation by gross anatomy
NOTE: Sacrifice the rats at the designated timepoint, 24 h and 14 d after surgery (Figure 1D).
6. Paraffin section and hematoxylin and eosin (HE) staining
7. Statistics
A total of 25 animals were used, 3 for control, 6 for 30 µL, 6 for 60 µL, and 10 for 100 µL blood injections. One rat that received a 100 µL injection of autologous blood (1/10) died within 24 hours after surgery.
Behavioral tests were conducted on Day 1, Day 3, Day 7 and Day 14 after surgery. The scores for the control group and blood-injection groups on different timepoints after surgery are presented in Table 2. The pontine hemorrhage caused neurological deficits like diminished corneal reflex and circling (Figure 3B,C). Injection of 100 µL blood also induced the myotonia (Figure 3A). The results of the balance beam test, limb placement test, and the modified Voetsch neuroscore revealed that the neurological function was decreased as the volume of pontine hemorrhage increased.
MRI scanning was performed 24 hours after surgery (Figure 4). In the blood-injection groups, on T2 sequence, hemorrhage was detected as a hypointense rim with an iso- to slightly hyperintense core in the basilar part of the pons. There was no hemorrhage detected by MRI in other brain areas (Figure 4). The volume of hemorrhage was increased as the injection volume of autologous blood increased.
Then rats were sacrificed at 24 hours and Day 14 after surgery, separately, and 2 mm thick sections were made (Figure 4). Hemorrhage was detected surrounding the injection site and distributing in the base of pons. There was slight edema around the hemorrhage in the 100 µL blood-injection group.
Some of the rats were sacrificed 3 days after surgery and paraffin-sectioned to do HE staining. Results showed that in the blood-injection groups, inflammatory cells enriched in the peri-hemorrhage zone (Figure 5C and F). The hemoglobin remains contained within intact red blood cells (Figure 5D and G).
Figure 1: Schematic diagrams of pontine hemorrhage model. (A) Autologous blood collection from tail vein. (B) The schematic diagram of drill location. (C) The schematic diagrams of injection location. (D) Experimental design. Please click here to view a larger version of this figure.
Figure 2: Instruments and procedure of surgery. (A) Anesthesia machine. (B) Surgical instruments. (C) Microdrill. (D) Micro-injection pump. (E) Stereotaxic apparatus. (F) A line marked in the middle of the skull. (G) Yellow arrow points to drill location. (H) Drainage of blood from the tail vein. (I) Transfer the blood into Eppendorf tube. (J) Aspirate the blood into Hamilton syringe. (K) Advance the Hamilton Syringe through the skull hole. (L) Injection process of autologous blood. Please click here to view a larger version of this figure.
Figure 3: Representative results of behavioral tests. (A) Myotonia in a rat injected 100 µL of autologous blood. (B) Diminished corneal reflex on the right side in a rat injected 60 µL of autologous blood. (C) Diminished corneal reflex in the bilateral sides in a rat injected 100 µL of autologous blood. (D) A rat received 60 µL of autologous blood circled to the contralateral side of lesion. (E) Results of balance beam test. (F) Results of the modified Voetsch neuroscore. (G) Results of limb placement test. Line means significant difference between the two groups (p < 0.05). Please click here to view a larger version of this figure.
Figure 4: Representative results of MRI scanning and gross anatomy. The MRI scanning (Upper) was performed 24 h after pontine hemorrhage surgery, then the rats were sacrificed and cut into 2 mm brain sections (Bottom). Please click here to view a larger version of this figure.
Figure 5: Representative results of HE staining. Brains were harvested from the rats injected 100 µL of blood 3 d after surgery. (A) The whole brain section. Low fields from (B) the normal pontine area, (C) peri-lesion zone and (D) hemorrhage core. High fields from (E) normal pontine area, (F) peri-lesion zone and (G) hemorrhage core. Scale bar was 100 µm. Please click here to view a larger version of this figure.
Figure S1: Representative results of gross anatomy on Day 14 after surgery. Please click here to download of this file.
Mild | Moderate | Massive | |
Total volume | 30 μL | 60 μL | 100 μL |
First injection | |||
Stereotactic coordinates | AP -9.0 mm; Lat 0; Vert -9.2 mm | ||
Volume | 10 μL | 10 μL | 10 μL |
Speed | 1 μL/min | 1 μL/min | 1 μL/min |
Interval time | 20 min | 20 min | 20 min |
Second injection | |||
Stereotactic coordinates | AP -9.0 mm; Lat 0; Vert -9.2 mm | AP -9.0 mm; Lat 0; Vert -9.0 mm | |
Volume | 20 μL | 50 μL | 90 μL |
Speed | 1 μL/min | 1 μL/min | 1 μL/min |
Before withdrawal of the needle | 10 min | 10 min | 10 min |
AP: anteroposterior position | |||
Lat: lateral | |||
Vert: vertical |
Table 1: Injection of autologous blood.
Rat Number | Day 1 | Day 3 | Day 7 | Day 14 |
The modified Voetsch neuroscore | ||||
30 μL-1 | 33 | 34 | 38 | 41 |
30 μL-2 | 30 | 35 | 37 | 41 |
30 μL-3 | 34 | 37 | 40 | 42 |
60 μL-4 | 27 | 30 | 36 | 38 |
60 μL-5 | 23 | 28 | 34 | 39 |
60 μL-6 | 26 | 29 | 35 | 39 |
100 μL-7 | 16 | 25 | 31 | 36 |
100 μL-8 | 13 | 22 | 29 | 37 |
100 μL-9 | 14 | 21 | 26 | 36 |
Sham-10 | 41 | 42 | 42 | 42 |
Sham-11 | 42 | 42 | 42 | 42 |
Sham-12 | 42 | 42 | 42 | 42 |
Balance beam test | ||||
30 μL-1 | 1 | 0 | 0 | 0 |
30 μL-2 | 1 | 1 | 0 | 0 |
30 μL-3 | 2 | 1 | 0 | 0 |
60 μL-4 | 3 | 2 | 0 | 0 |
60 μL-5 | 4 | 2 | 0 | 0 |
60 μL-6 | 3 | 2 | 1 | 0 |
100 μL-7 | 5 | 4 | 3 | 1 |
100 μL-8 | 5 | 4 | 2 | 1 |
100 μL-9 | 4 | 4 | 2 | 1 |
Sham-10 | 0 | 0 | 0 | 0 |
Sham-11 | 0 | 0 | 0 | 0 |
Sham-12 | 0 | 0 | 0 | 0 |
Limb placement test | ||||
30 μL-1 | 11 | 12 | 12 | 12 |
30 μL-2 | 10 | 11 | 12 | 12 |
30 μL-3 | 10 | 11 | 12 | 12 |
60 μL-4 | 9 | 11 | 12 | 12 |
60 μL-5 | 8 | 9 | 9 | 11 |
60 μL-6 | 8 | 9 | 10 | 11 |
100 μL-7 | 4 | 5 | 9 | 11 |
100 μL-8 | 3 | 4 | 8 | 10 |
100 μL-9 | 2 | 4 | 7 | 8 |
Sham-10 | 11 | 12 | 12 | 12 |
Sham-11 | 12 | 12 | 12 | 12 |
Sham-12 | 12 | 12 | 12 | 12 |
Table 2: Results of behavioral tests.
In the present study, we provided a protocol to generate a massive pontine hemorrhage rat model. This model can be used for the research on the pathophysiological mechanism and prognosis of massive pontine hemorrhage.
Throughout the experiment, 25 rats were used, of which only one died. The verification of MRI, gross anatomy, and the HE staining indicated that this method had a very low mortality rate and a high success rate. To establish massive pontine hemorrhage model, two problems must be solved, the injected autologous blood tends to leak into the subarachnoid space and flow back to the fourth ventricle along the needle tract. The existing double-injection moderate (60 µL autologous blood in total) pontine hemorrhage model resolved the first problem, barely any blood flowed into the subarachnoid space. However, there was still a small amount of blood backflow. In the present study, several strategies were applied to optimize the existing double-injection method to make it possible to inject a larger amount of 100 µL autologous blood without backflow. First, two different injection spots instead of one were employed. Second, heparin was used to flush the syringe with minimal residual to reduce the dosage, in order to only protect the blood from coagulating during the injection process, but not enough to promote leakage and backflow after the injection. Third, the injection time was long and injection speed was slow, 1 µL/min. Moreover, only a small amount of autologous blood was injected the first time, while the second injection was performed 20 minutes later. Afterwards, the needle was withdrawn only after 10 minutes, and this procedure was conducted extremely slowly. Using this method, there was barely any blood flowing into the subarachnoid space or fourth ventricle in the rats injected with 30 µL or 60 µL of autologous blood, but there was still a small amount of backflow in the rats injected with 100 µL. Extending the time before removing the needle could solve this problem.
Behavioral tests were performed on Day 1, Day 3, Day 7 and Day 14 after modeling, including balance beam test, limb placement test, and the modified Voetsch neuroscore. On the first day after surgery, almost all of the rats in the blood-injection groups showed circling behavior (i.e., turning left or right), accompanied by disappearance of unilateral or bilateral corneal reflex. Although autologous blood was injected in the midline of the pontine, it was unevenly distributed in the two sides of the brain. This seemed to be the reason for the different performance in behavioral tests. The activities and reactions of the rats injected 30 µL or 60 µL of autologous blood slowed closer to normal. In the rats injected 100 µL of blood, the sensorimotor functions were significantly weakened and the response was poor. Muscle rigidity appeared in some rats in the resting state. On Day 1, Day 3 and Day 7, there were obvious differences between rats injected 30 µL, 60 µL or 100 µL of autologous blood and the rats in the control group in the modified Voetsch neuroscore. In the balance beam test and limb place test, there was no significant differences between the rats injected 30 µL or 60 µL of blood and the rats in control group at any time points. However, the results of the balance beam test and limb placement test in rats injected 100 µL of autologous blood were significantly different when compared with the control group on Day 1 and Day 3. The possible reason could be that there are fewer evaluation items in the balance beam test and the limb placement test compared to the modified Voetsch neuroscore, which are not sensitive enough to discover subtle neurological deficits. It is inappropriate to use these two methods to evaluate behavior in hemorrhage models with mild symptoms, but they are applicable in the massive pontine hemorrhage model. Overall, the modified Voetsch neuroscore turned out to be more suitable for comprehensively and accurately assessing the neurological functions in different pontine hemorrhage models.
There are several advantages of this method. Based on the previous double-injection method, the second injection location was changed and the dosage of heparin was adjusted to avoid the leakage and backflow in the mild (30 µL) and moderate (60 µL) pontine hemorrhage model. Even in the massive (100 µL) pontine hemorrhage model, the backflow was very limited, and occurred only in a small number of rats. This method can be easily performed with a high success rate and a low death rate. Moreover, the experimental pontine hemorrhage can be observed during a long period, at least 14 days after modeling, which is conducive to investigating the entire disease development and effects of treatments. The major advance of this model was that it mimicked the symptoms of patients with pontine hemorrhage. Clinically, massive pontine hemorrhage results in severe neurological deficits, while previous pontine hemorrhage models only developed relatively small hemorrhagic volume with mild symptoms. The massive pontine hemorrhage in this model distributed in the bilateral pons, which is similar with hemorrhage distribution in pontine hemorrhage patients. In previous experimental pontine hemorrhage models, the hemorrhage only located in the unilateral pons9.
However, there are also some limitations of this method. First, pontine hemorrhage in this study was caused by injection of blood, partially heparinized during transition, which might influence the blood coagulation or even homeostasis in the surrounding pons. Second, this model requires special equipment, such as stereotaxic apparatus and injection pump. Third, this model cannot mimic spontaneous hemorrhage.
In conclusion, this study provided a method to create an experimental acute massive pontine hemorrhage model in the rat, which could promote new mechanical and therapeutic research in this field.
The authors have nothing to disclose.
This study was financially supported by the National Science Foundation of China (81471181 and 81870933) and the Opening Lab Program of Guangzhou Medical University (0506308) to Y Jiang, and by the National Science Foundation of China (81701471) and the Scientific Program of Guangzhou Municipal Health Commission (20191A011083) to Z Qiu, and by the National Science Foundation of China (81501009) to L Wu.
100ml Saline solution | Guangdong yixiang | 191222201 C1 | Preparing heparin diluent |
100μl Microinjector | Shanghai Gaoge | Injection of autologous blood | |
1ml Syringe | Jiangsu Zhiyu | 20191014 | Withdraw autologous blood from the tail vein |
75% Alcohol | Shandong Lierkang | Disinfection of rat tail | |
Adhesive tape | Shanghai Jinzhong | Surgicl instruments | |
Animal anesthesia system | RWD | R510-31S-6 | Inducing and maintaining anesthesia |
Balance beam | Jiangsu Saiangsi | For neurological deficit scores | |
Blades | Shanghai Feiying | 74-C | For gross anatomy |
Bone cement | Shanghai Xinshiji | 20180306 | Surgicl instruments |
Brain tank | Shenzhen LEIYEA | For gross anatomy | |
Butorphanol tartrate | Jiangsu Hengrui | For pain management | |
Electric cranial drill | Nanjing Darwin biotechnology | 20180090018 | Making a burr hole on the skull |
EP tube | Nantong Surui | Transfer autologous blood | |
Erythromycin eye cream | Yunnan pharmacy | Eyes protection | |
HE dye liquor | Solarbio | G1120 | For HE staining |
Heating pad | Dangerous Jungle | JR01 | Keeping warm |
Heparin sodium injection | Chengdu Haitong Pharmacal Company | 190701 | Preparing heparin diluent |
IndoPhors | Guoyao of China | Sterilization | |
Isoflurane | RWD | 20080701 | Inducing and maintaining anesthesia |
Light dark box | Jiangsu Saiangsi | For neurological deficit scores | |
Micro-injection pump | Baoding Leifu | TFD03-01-C | Injection of autologous blood |
MRI system | Philips | Confirmation of infarction in vivo | |
Needle holder | Shanghai Jinzhong | J32020 | Surgicl instruments |
Penicilin | Guoyao of China | Infection Prevention | |
Q-tips | Jiangxi Songhe | Surgicl instruments | |
Scalp heedle | Jiangxi Hongda | 20200313 | Withdraw autologous blood from the tail vein |
Scalpel | Shanghai Kaiyuan | 170902 | Surgicl instruments |
Shearing scissors | Shanghai Jinzhong | Y00040 | Surgicl instruments |
Stereotaxic apparatus | RWD | 900-00001-00 | for surgical positioning |
Surgical towel | Xinxiang Huakangweicai | 20070601 | Surgicl instruments |
Suture needle | Shanghai Jinzhong | Surgicl instruments | |
Suture scissors | Shanghai Jinzhong | J25041 | Surgicl instruments |
Tissue holding forcepts | Shanghai Jinzhong | J31080 | Surgicl instruments |