Preparation of Laboratory Animal Models of S-TBI and Its Clinical Significance

Lu Shou Long^1*, Ying Qi¹, Zhang Feng Lin¹, Xu Xiao Long¹, Tang Yin², Jiao Yong², Wang Quan², Wang Xiao Xi³, Zhang Xiao Li³ & Lu Ning⁴

¹Department of Neurosurgery, Shanghai Naval Hospital, China
²Animal Laboratory and Medical Information, Department of Naval Medical Research Institute, China
³Department of Pathology, Naval Hospital, China
⁴Information Center of Shanghai Research Institute of Sinopec, China

*Correspondence to: Dr. Lu Shou Long, Department of Neurosurgery of No.411 Naval Hospital, China.

Copyright © 2019 Dr. Lu Shou Long, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Traumatic brain injury (TBI), especially severe traumatic injury (S-TBI) is a difficult problem the medical circle is now facing, and the focus of research [1]. To enhance the level of treatment of S-TBI the author explored, in this paper, the replication of S-TBI models. There have been many methods of replicating TBI models through animal experiments, but few have been reported on animal experiments similar to human severe traumatic brain injury [2]. Through a series of animal experiments the author found the optimum trauma-causing impact force for creating S-TBI models in experimental animals, and discovered the correlation of pathological changes to clinical manifestation of brain trauma through post-trauma observation at different time points and reading of a lot of pathological sections. These findings have a reference value to the improvement of clinical diagnosis and treatment [3]. The author also made a study of the manifestation of closed decompression and open decompression in animal models and a comparison of the pathological changes, and from them gained some useful enlightenment.

Materials and Methods
The experimental animals were provided by Shanghai Sippr BK Laboratory Animal Co. Ltd. They were SD/SPF white rats, totalling 80, all male, and each weighing 225-337g. The rats were divided into groups at random according to the force of percussion.

An improved traditional free fall apparatus was used to cause brain injury. It consisted of a brace, a weight, a guide and a base (See Fig. 1).

1.Percussion Weight, 2. Lift Line, 3. Guide, 4. Bracket, 5. Wooden Board with Sponge

Rat models of closed brain injury were given intraperitoneal anesthesia with 10% chloral hydrate in 0.35g/ kg. A longitudinal incision of about 3cm in length was then made on the scalp after disinfection along the median line to expose bilateral vaults of skull. Under anaesthesia the exposed part of skull was impacted by free fall with different weights and at different heights. The scalp was sutured immediately after percussion. The rats were then placed in the cage for observation and fed in the routine way.

The percussion weights were 50g, 100g, 250g, and 500g respectively, and the heights were 20cm, 30cm, 37cm, 40cm, and 50cm respectively.

The percussion causing brain injury of experimental animals was the result of the weight of the percussion mass multiplied by the height of its departure point in g/cm [4].

The animals were divided into groups as shown in the following table.

*The three in non-injury group were the control group.

Of the 1000g/cm group 6 rats were chosen at random, and of the 10000g/cm group 6 were also chosen at random. Closed decompression and open decompression were performed in a ratio of equality. In rats receiving closed decompression, the two-side fornices of skull were removed after percussion with a grinding drill, the bilateral dura mater of brain was incised in line, and the scalp sutured. While in rats given open decompression the two-side fornices of skull were removed, the bilateral dura mater of brain incised in line to expose the surface of wound and aseptic normal saline gauze was applied to it.

Intra-peritoneal anesthesia was performed with 10% chloral hydrate (0.35g/kg) in rats of various groups and brain was taken 4h, 6h, 24h, 72h(3d), 168h(7d), 288h(12d) after injury, and was put into the formaldehyde solution and fixed above 24 hours.

For collection of material and observation of pathological sections, a sagittal piece about 5mm thick including the cerebrum, brain stem and cerebellum was taken 1-2mm to the outer side of the median line, and embedded in paraffin. Serial sections 1μm thick were made and HE stained. The pathological sections were read in sequence of parietal lobe, frontal lobe, base of skull, ventricles of brain, and if any lesions were found they were photographed in sequence of low, middle and high magnification (L:10x4, M:10x10, and H:10x40) [5].

Result
There was a significant difference in reaction and percussion among laboratory rats after percussion by heavy weights under anaesthesia [5]. When the percussion was 1000g/cm, only convulsion of left or right lower limb lasting about one second was found; when the percussion was 3000g/cm, the palpitation and respiration of the rats receiving impact were markedly increased; with percussion at 4000g/cm the rats developed sudden general convulsion lasting 1-2 seconds, increased respiration and palpitation, and about 3 seconds of convulsion of left or right lower limb; at 5000g/cm, blood stain was seen on the surface of the percussion weight and the rats were found to have sudden convulsion of increased extent; at 10000g/cm, a 2-second large-extent general convulsion and jump was found in rats, and was accompanied by bending of back in one case; and with the percussion over 10000g/cm, all the rats had nose and mouth bleeding followed immediately by death.

In the rats of 10000g/cm group, when brain was collected 7 days later, old extradural hematoma and subdural hematoma were both found; the skull showed looseness and brittleness, and fragmentation of brain tissue was seen.

In the rats surviving head percussion, the time taken to regain consciousness from anaesthesia, the frequency of activity, and the appetite were correlative to the percussion force [6]. The time taken to regain consciousness from anaesthesia was generally over an hour. One rat had orientation signs, for example, rotation and crawling to the right. Another rat (receiving a percussion of 10000g/cm), after it came to, developed listlessness, refusal of food and water, and blepharoptosis, and died 2 days later.

The frequency of activity in the rats receiving open decompression was higher than those receiving closed decompression. The open wound on the top of the head healed up by itself 4 days later.

652 images were made for observation of pathological sections.

The image of cerebral cortex of normal non-injured rats showed a distinct arachnoid and a good continuity, clear subarachnoid cavity, and uniform distribution of molecular layer, granular layer, pyramidal cell layer and glia cells in the cortex (I-IV) (Fig. 2). The four ventricles of brain and the choroid plexus were in regular shape (Fig. 3).

The rats after head injury had diffuse injury involving the whole brain, and in some area the injury was severe. It was shown as marked cerebral edema, swelling of nerve cell, and asymmetrical degenerative necrosis of nucleus at the site of injury (Fig. 4). Neurotropism of pyramidal cell was found after injury (Fig. 5). Hemorrhage was seen in the region of brain parenchyma and local hematoma formed (Fig. 6). Loose and spongiform brain tissue, massive vacuolization, and disturbance of alignment, fragmentation, and distortion and deformation of nerve fibre were also observed [7]. Traumatic subarachnoid hemorrhage was shown (Fig. 8). There was marked expansion of ventricles of brain with traumatic injury (Fig. 9). The cerebellum displayed petechial hemorrhage, and swelling, degeneration and necrosis of Purkinje`s cell, and neurotropism of small glia cells surrounding Purkinje`s cells (Fig. 10). Hemorrhage of micrangium in the zone of brain stem, loose and spongiform brain tissue, and pyknosis of partial nerve cells were found (Fig. 11).

In rats with closed decompression, there was massive edema in the cerebral cortex region (Fig. 12) and marked vacuolar degeneration after closed decompression (Fig. 13).

In rata with open decompression, superficial edema in small extents was seen in the cerebral cortex region (Fig. 14). There was vacuolar degeneration after open decompression, but in a light extent (Fig. 15).

Rats receiving a percussion of 1000g/cm had marked expansion of ventricles of brain and fragmentation of partial cerebellum tissue 4 hours after injury (Fig. 16). 12 days after percussion, the cerebral cortex showed massive proliferation of glial fibre in some areas (Fig. 17).

Discussion
To simulate human head trauma, free fall impact was adopted to create a condition more like the actuality of human head injury. As the scalp of the rat was loose and slippery, it was used to make a 3cm long incision along the median line of the head after anesthesia so as to prevent inaccuracy in the location receiving percussion, which might be caused by the move of scalp, and obtain a direct traumatic result. A traction line was secured to the top of the percussion weight to prevent secondary percussion. The result achieved by this method of giving percussion to the head of the animals in this experiment was similar to that in accelerated brain injury, and it was controllable, replicable, simple and easy to carry out [7].

This animal experiment of brain injury was divided into 3 stages and the percussion weight and height were adjusted progressively. From the result of the experiment it could be concluded that 10000g/cm was the critical impact. Any percussion over 10000g/cm could cause immediate death of the laboratory rats. The cause of death was the excess force exerted upon the head of the rats resulting in bleeding of mouth and nose, airway obstruction, and suffocation. It was also an important cause of death that this percussion was beyond the limit of endurance of the brain tissue thus resulting in instantaneous disturbance of nerve function of hypothalamus. This experiment could give us some inspiration on the clinical work of neurosurgery. When the percussion on the head of the rats reached one that caused trauma, that is, 10000g/cm, local traumatic reaction of brain could occur not only at the site of percussion, but in the depth of brain including the cerebellum and the brain stem zone contusion reaction could also occur. Therefore, clinically S-TBI should be considered to be general brain injury. It could explain why sometimes the patient, though with the edema removed, would still remain in coma and his condition was still deteriorated.

Like the human beings and other organisms the rodent white rats also had the function of self-repair after their brain was injured by percussion and it was observed from pathological sections that edema was reduced in scope and bleeding was absorbed. In rats receiving open decompression the wound was scabbed and healed up 4 days later.

After the brain was injured, the occurrence of neurotropism of the injured pyramidal cells accompanied by degeneration and necrosis was an important factor that caused signs of orientation in clinical patients. Diffuse axon injury was a sort of rather severe brain injury and the fragmentation of a large amount of nerve fibres, edema, distortion, degeneration and demyelination were one of the causes of long-term coma and intractable cerebral edema in the patients [8].

Traumatic subarachnoid hemorrhage could increase the intracranial pressure and cause headache in the patients after trauma. Within a few days of arachnoid hemorrhage spasm of blood vessel of brain would occur in 60-70% patients and was accompanied with cerebral ischemia in half of the patients, so ventricular drainage or drainage of lumber cistern under monitoring of intracranial pressure in early time after trauma could bring a preventive effect [9].

The ventricles of brain might be enlarged or reduced after traumatic brain injury. The enlargement was due to intraventricular hemorrhage and obstruction of cerebrospinal fluid passage, while the shrinkage was possibly due to cerebral edema and the squeeze of lateral ventricle resulting from brain swelling [10].

From the pathological sections it could be seen that there was a difference in edema of brain cortex at the site of wound between closed decompression and open decompression. The edema in the former (closed decompression) involved a larger and deeper area, while that in the later (open decompression) involved a smaller and superficial area. Vacuolar degeneration was in a lighter extent in the later comparing with the former. This was because the high cranial pressure resulting from brain injury was released toward the outside after open decompression, and with the hemic cerebrospinal fluid overflowing the intracranial pressure decreased. It thus helped the experimental rats to tide over cerebral edema and brain swelling occurring after trauma and the recovery of brain injury.

TBI (trauma brain injury) includes primary injury and secondary injury. In primary brain injury, various external forces might cause the impulsive reaction of the blood vessel and nerve tissue, e.g. intracranial hemorrhage, intracranial hypertension, decrease of cerebral blood flow, cerebral edema and diffuse axon injury (DAI). While secondary brain injury could lead to a series of complex post-traumatic pathological and physiological reaction. And in severe traumatic brain injury (S-TBI) the above-mentioned reaction was more obvious and the prognosis more severe. However, cerebral trauma is in essence the reaction of organism to trauma, but it occurred only in the nerve system, a particular anatomic site. Knowing this point the clinical doctors and researchers will be able to take the right methods of treatment. The most obvious post-traumatic reaction is intracranial hypertension, and now the commonly used decompression with removal of large bone flap is effective to a certain degree, but not fully effective, especially for severe TBI. Open decompression has shown an important and effective way and the results of pathological observation described above are the evidence.

Prospect
TBI animal models can be used in the serial studies of brain injury. Since the death rate and maim rate are extremely high in severe traumatic brain injury, it should be the focus of future studies. By creating laboratory animal models of severe TBI and selecting large animals, e.g. rabbits, cats and dogs, a greater similarity to human traumatic reaction may be obtained.

This method of preparing animal models may contribute to the change of traditional pattern of treatment and way of thinking.

Bibliography

1. Zhang Xiang (2001). Basis of Severe Brain Injury and Its Clinical Treatment. PLA Medical Journal., 2(8), 7.
2. Song Yang, Han Yi Sheng, Bi Long, et al. (2009). The Establishment of Laboratory Rat Models of Traumatic Brain Injury and Its Appraisal System. China J Neurosurg Dis Res., 8(6), 521-524.
3. Li Shang Xun, Wang Bo Wei, Liu Dan, et al. (2011). Progress in the Study of Animal Model of Traumatic Brain Injury. Journal of Forensic Medicine, 27(4), 286-289.
4. Duan Hu Bin, Hao Chun Yan, Liu Cui Ming, et al. (2013). The Correlation of Blood Bradykinin with Expression of P-Substance of Brain Tissue in the Trauma Area in Rats after Traumatic Brain Injury. Chinese Journal of Integrative Medicine on Cardio-Cerebro Vascular Disease, 11(7), 845-846.
5. Finnie, J. W. (1993). Pathology of Experimental Traumatic Cranio-Cerebral Missile Injury. [J] Comp Pathol., 108(1), 93-101.
6. Cernek I. Animal Models of Head Trauma. [J] Neuro RX., 2(3), 410-422, 2005.
7. Marmarous, A., Foda M. A., Van den Brink W, et al. New Model of Diffuse Brain Injury in Rats. Part 1: Pathophysiology and Biomechanics. [J] Neurosurg., 80(2), 291-300.
8. Gennarell, T. A., Thibault, L. E., Adams, J. H., et al. (1982). Diffuse Axonal Injury and Traumatic Coma in the Primate. [J] Ann Neurol., 12(6), 564-574.
9. Edlow, J. A. (2005). Diagnosis of Subarachnoid Hemorrhage. Neurocrit Care, 2, 99-109.
10. Licata, C., Cristofori, L., Gambin, R., et al. (2001). Post-Traumatic Hydrophalus. J Neurosurg Sci., 45(3), 141.