This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.
Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.
Traumatic brain injury (TBI) is a general term for the acquired injury to the brain due to either indirect mechanical forces (rotational acceleration/deceleration or contra-coup) from blows to the head or direct damage from objects or blast waves. TBI has been estimated to be the cause of roughly 9% of worldwide deaths and observed in an estimated 50 million cases per year1,2. A 2017 report from the Centers for Disease Control and Prevention estimated that in 2013, there were a total of 2.8 million hospital visits and deaths due to TBI in the United States3. Many milder TBIs go unreported every year. Serious TBI can lead to lifelong impairment of cognition, motor function, and overall quality of life. The consequences of mild TBI, especially repetitive sport-related TBI, have been only recently appreciated for their insidious health effects4,5.
Preclinical modeling is a vital component of developing new mechanistic insights and potential restorative therapy for TBI. The controlled cortical impact (CCI) model of TBI is an open-head model of mechanical contusion injury to the cortex. The impact parameters can be modified to produce CCI injuries that range from mild to severe6. CCI injuries are focal rather than diffuse, as seen with other closed head models of TBI. CCI can be performed to induce a unilateral injury, such that the contralateral cortex can serve as an internal comparator. This protocol demonstrates the characteristics of a mild CCI to a portion of the cortex that encompasses primary somatosensory and motor regions. This cortical area was chosen for its involvement in sensorimotor behaviors for which numerous behavior tests can detect injury-induced deficits7. Behavioral improvements due to therapeutic interventions for TBI can be detected, as well.
A hallmark of TBI is widespread neural dysfunction in the injured region. Injured neurons undergo cell death, and neuronal network connectivity is disrupted8,9. TBI disrupts recruitment of endogenous stem cells, which leads to further downstream behavior deficits10,11. Transplantation of neural stem cells and stem cell-derived cells has been explored as a possibility to restore function in the injured brain. In addition to the potential to restore damaged neural circuitry, transplanted cells exert paracrine effects that promote neuronal survival and functional recovery from TBI12. A variety of cell types have been transplanted preclinically to evaluate their restorative potential in models of neurologic disorders13,14,15. The recent popularization of induced pluripotent stem cell technology16 has facilitated the development of numerous human stem cell lines for experimental use. Preclinical testing with hiPSC-derived cells is an important first step to characterizing a given cell line’s potential therapeutic efficacy against human diseases. This laboratory has developed protocols for differentiating hiPSCs to neural phenotypes17 in pursuit of transplantable cells to aid recovery from traumatic brain injury.
Experiments in this protocol use a unilateral CCI to induce TBI to the left somatosensory and motor cortex of adult mice. A mild CCI injury results in a sustained functional deficit in the right forepaw that is used to track the effects of hiPSC-derived neural cell engraftment on functional recovery. Forepaw sensorimotor testing in this protocol was adapted from the methodology established by Bouet and colleagues18 and demonstrated previously by Fleming and colleagues19. This protocol outlines a complete workflow for performing an experimental brain injury, therapeutic transplantation of hiPS cells, and behavioral and histologic analysis of experimental outcome measures.
All experiments described in this protocol were reviewed and approved by the Uniformed Services University Animal Care and Use Committee.
1. Craniectomy and controlled cortical impact
2. Stereotaxic transplantation of cell suspension
3. Adhesive tape removal test of sensorimotor integration
4. Diaminobenzidine (DAB) immunohistochemical analysis of graft survival and injury pathology
Craniectomy surgery facilitates experimental brain injury and therapeutic cell transplantation: the controlled cortical impact model of brain injury and subsequent cell transplantation therapy require careful removal of the overlying skull. The craniectomy may be performed on any dorsal surface of the skull to permit manipulations to the brain region of interest. The diagram in Figure 1 depicts a 5 mm diameter craniectomy schematic to uncover primary somatosensory and motor cortices (Figure 1A). At 24 h after craniectomy, a second surgery was performed to inject human iPSC-derived neural cell suspension into deep layers of the cortex (Figure 1B). Some cerebral edema is normal on the first day following craniectomy, and particularly after CCI. However, cerebral vasculature sparing during all phases of this procedure is crucial for survival of the cortex. Figure 2 illustrates the cell transplantation procedure in a mouse with minimal cerebral herniation, minimal bleeding, and extensive cortical vascularization. These features are good prognostic indicators of a successful surgery.
Adhesive tape removal testing reveals sensorimotor deficits after unilateral brain injury: the parameters of the brain injury model described above were predicted to affect forelimb sensory and motor function. The adhesive tape removal test was chosen to evaluate the severity of forelimb functional deficits, and the potential therapeutic benefits of cell transplantation. Mice were trained on the testing procedure for 5 days, then allowed to rest for two days prior to baseline behavior testing. Surgeries were performed on the day following baseline testing. Behavior tests in this study were performed on postoperative days 1, 3, 5, 7, 10, 14, 21, 28, 35, and 42. Figure 3 shows results from a pilot experiment in which forelimb function in mice with craniectomy alone (sham) and with CCI injury were compared to forelimb function in naïve mice (n = 11 naïve, 12 sham, 11 CCI). Mice that underwent surgery exhibited transient increased latencies to notice adhesive stimuli for 1-3 days immediately after surgery (Figure 3A,B). Mice showed transient postoperative deficits in adhesive removal from the ipsilateral forepaw as well (Figure 3C). However, mice that underwent CCI exhibited significant deficits in motor performance in the forepaw contralateral to injury compared to naïve mice out to postoperative day 28 (Figure 3D). These data also describe the unexpected severity of sensorimotor loss in craniotomized mice without CCI, indicating that surgical craniectomy to this area also induces TBI-related neurofunctional deficits.
Immunodetection of human induced pluripotent stem cell (iPSC)-derived cell grafts in mouse brain sections: experiments were performed to determine whether human iPSC-derived neural cells would survive long-term transplantation in the mouse brain. Human neural stem cells (NSCs) derived from iPSCs were differentiated into either immature neurons or astrocytes in vitro using established methods17. Transplants of each of the three neural cell phenotypes were tested in our CCI model of traumatic brain injury using the procedure described above and depicted in Figure 2. The mice were euthanized for histologic analysis at 7 days after transplantation. Mouse brain sections were immunostained for the human nuclear antigen (hNA). Human cell grafts could be clearly distinguished from host tissue in sham surgery and CCI brains (Figure 4). Astrocyte grafts (n = 3 sham, 2 CCI) showed poor survival compared to NSCs (n = 12 sham, 15 CCI) and neurons (n = 11 sham, 10 CCI), and were not considered for future experiments.
Figure 1: Coordinate parameters of surgical manipulations. Cartoon depictions of mouse brain regions of interest. Red circles indicate a ~5 mm diameter craniectomy. A red cross indicates the craniectomy central point 2 mm lateral to bregma. (A) The shaded region of cerebral cortex in the upper diagram is affected by mild CCI when a craniectomy is performed as shown in lower diagram. (B) The blue arrow in upper diagram indicates the approximate location of cell injections at 1.4 mm depth from cortical surface. The blue cross in the lower diagram indicates the placement of cell injection 2 mm lateral and 1 mm posterior to bregma. Please click here to view a larger version of this figure.
Figure 2: Intraoperative monitoring of cell suspension injection. Photograph taken through a long working distance microscope during intraparenchymal cell injection. Anatomic features are annotated for clarity. The scalp partially obscures the surgery site to minimize dehydration during the procedure. Minor bleeding may occur during needle penetration as shown, which is not cause for concern if large cortical vessels remain intact. Please click here to view a larger version of this figure.
Figure 3: Behavioral evaluation of sensorimotor integration after brain injury. Mice that underwent craniectomy and CCI were compared to naïve controls and to mice that underwent only sham surgery (n = 11 naïve, 12 sham, 11 CCI). Data are presented as group mean latencies, with error bars indicating SEM. (A) Mice that underwent CCI exhibited increased latency to recognize adhesive stimuli applied to the ipsilateral forepaw on the first postoperative day. (B) Mice that underwent craniectomy or CCI exhibited substantially increased latency to notice adhesive stimuli applied to the contralateral forepaw on postoperative days 1 and 3. (C) Mice that underwent craniectomy or CCI exhibited substantially increased latency to remove adhesive stimuli from the ipsilateral forepaw on postoperative days 1 and 3. (D) Mice that underwent craniectomy or CCI exhibited substantially increased latency to remove adhesive stimuli from the contralateral forepaw on postoperative days 1-5. Motor deficits in mice with CCI persisted strongly for 28 days after injury. Please click here to view a larger version of this figure.
Figure 4: DAB immunohistochemistry for human cell grafts in mouse brains. Human iPSCs were differentiated into neural stem cells (NSCs), neurons, or astrocytes in vitro. Cell cultures were transplanted into mouse brains with or without CCI. Mice were euthanized for histologic analysis seven days after cell transplantation. Micrographs depict representative results of human nuclear antigen staining. Black insets depict markers for stereologic quantification of cell numbers (cyan) and graft volume (red). Please click here to view a larger version of this figure.
Mild CCI as a model system for testing experimental regenerative therapy
The CCI model is a valuable tool for investigating mechanisms of tissue dysfunction after mechanical injury to the cortex. The tunability of the injury parameters is an attractive feature of this model. Altering the Z depth of impact, the velocity, or dwell time can increase or decrease severity of the injury as desired by the investigator10,25. The mild CCI model of contusive brain injury, when performed correctly, should cause modest cortical cell death and minimal cavitation. Craniectomy and skull flap removal must be performed with great care. Excessive downward force applied while drilling the craniectomy trench can cause cortical injury due to heating and vibration. Mechanical disruption of the dura mater during skull flap removal almost uniformly predicts severe cortical injury. Disruption of major cortical blood vessels is likely to result in excessive lesioning of the cortex and is grounds for excluding the animal from the experiment. Unfortunately, the signs of an exacerbated injury can be subtle on the day after surgery. Neither edema nor small cortical vessel rupture are necessarily negative indicators. Hematomas and abnormal coloration due to ischemia are clearer indicators of surgical complication. Documenting intraoperative events and correlating complications with histopathologic outcomes are crucial to refining good craniectomy technique.
It must be noted that mTBI modeling in animals comes with certain caveats. There are numerous preclinical models of mTBI other than the model presented here. Experimental TBI can be induced through mechanical forces, blast waves through air, or a combination of these forces26,27,28. Mild to severe injuries are judged by a combination of histopathological and behavioral outcomes (reviewed in Petraglia29 and Siebold30). Behavior deficits in rodents can resolve within days to weeks31 whereas human mTBI patients’ deficits can persist for months in the form of post-concussive syndrome32. Although no single model is a complete analog for clinical mTBI, preclinical testing reveals physiologic mechanisms that cannot be assessed in the human condition.
The most important steps in the cell transplantation procedure are the handling and injection of the cell suspension. Rough handling causes cell lysis, leading to the release of sticky genomic DNA and aggregation of the surviving suspended cells. The needle tip diameter must be wide enough to permit smooth outflow of the suspension; restricted flow causes discontinuous delivery as the suspension sediments inside the needle. When following this protocol and avoiding the pitfalls discussed here, robust grafts were visible after 7-day survival times (Figure 4). Long term graft survival in a preclinical model is key to determining potential therapeutic efficacy of this approach.
Application of the adhesive tape removal test to contusive brain injury modeling
The adhesive removal test reveals positive neurologic deficits in terms of increased latency to remove the adhesive stimuli. The main potential drawback to this test is the likelihood of inhibited performance due to factors not related to injury. Handling stress can reduce mouse exploratory behavior33, so it is crucial that the animals undergo extensive restraint acclimation prior to baseline data collection. It is important to remove water supplies at least 30 min prior to testing in order to mitigate urination-related freezing events. Finally, the testing apparatus must be cleaned often as animals can become distracted into investigatory sniffing of odor cues from unfamiliar animals.
Results presented here show significant forepaw motor deficits contralateral to mild CCI up to 28 days after injury. By contrast, concurrent testing using the cylinder test34 and accelerating rotarod3 showed injury-induced functional deficits that resolved within 5–10 days (data not shown). Adhesive tape removal has been used in a variety of experiments evaluating unilateral deficits in sensorimotor integrative behavior7,35. The test was also recently used to assess motor function recovery following regenerative intervention for cervical spinal cord injury36. The dynamic range of performance on this test can be tuned for latency or species variation by selecting adhesives of different strength. Here, it is suggested that 3M electrical tape is an optimal stimulus for mice based on availability, durability, and substantially increased latency to remove stimuli following mild CCI. Although the preliminary experiments shown here do not combine cell transplantation and behavior testing, ongoing experiments in our laboratory will assess transplant survival and concurrent effects on sensorimotor behavioral recovery from brain injury at 56 days after transplantation.
Refinement to DAB immunodetection of human cell grafts
DAB immunohistochemistry was chosen in order to produce strong, persistent labeling of human cells in mouse tissue for subsequent stereologic quantification. Pretreatment with hydrogen peroxide is crucial for reducing nonspecific staining in brain tissue due to endogenous peroxidases in erythrocytes. Nonspecific staining in these experiments was further reduced using a mouse IgG neutralization kit. This kit uses proprietary chemicals to reduce antigenicity of endogenous mouse IgGs, which can extravasate into brain tissue after TBI37. Early attempts employed a combination of mouse anti-hNA primary antibody, biotin-conjugated secondary antibodies, and streptavidin-HRP conjugate tertiary labeling using the Vector Laboratories ABC kit. The ABC conjugate exhibited extensive nonspecific DAB staining in the cortex ipsilateral to the craniectomy (data not shown). Subsequent staining trials employed secondary antibodies directly conjugated with HRP. This modified protocol produces high-resolution nuclear staining with greatly reduced background for all hiPS-derived cell types and surgery conditions (Figure 4). Unpublished experiments from this laboratory using this modified DAB staining technique detect hNA-positive cells in mouse brains 56 days after mild CCI. Overall, a binary antibody labeling procedure saved time and produced clearer immunostaining compared to the traditional tertiary labeling ABC kit procedure. This protocol could be useful for other preclinical studies of human cells transplanted into the mouse central nervous system.
The authors have nothing to disclose.
This work was supported by a grant from the Center for Neuroscience and Regenerative Medicine (CNRM, grant number G170244014). We appreciate the assistance of Mahima Dewan and Clara Selbrede in adhesive removal pilot studies. Kryslaine Radomski performed preliminary brain injury and cell transplantation surgeries. Amanda Fu and Laura Tucker of the USU CNRM Preclinical Studies core laboratory provided valuable advice on animal surgeries and behavior testing, respectively.
1 ml syringes | Becton Dickinson (BD) | 309659 | |
1.7 ml flip top test tubes | Denville | C2170 | |
10 microliter syringe | Hamilton | 7635-01 | |
25G Precision Glide syringe needles | Becton Dickinson (BD) | 305122 | |
70% ethanol | Product of choice; varies by region | ||
acetaminophen oral suspension | Tylenol (Children's) | Dilute to 1 mg/ml in water | |
anesthetic vaporizer | Vetland | 521-11-22 | |
animal handling cloth | Purchase from department store | ||
Betadine | Purdue Products | NDC-67618-151-32 | |
compressed oxygen | Product of choice; varies by region | ||
cyclosporine A | Sigma-Aldrich | 30024-100mg | |
DAB staining kit | Vector Laboratories | SK-4100 | |
dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418-500ml | |
DMEM | Invitrogen (ThermoFisher) | A14430-01 | |
donkey anti-mouse IgG antibody, HRP conjugated | Jackson ImmunoResearch | 715-035-151 | |
electrical tape | 3M Corporation | Purchase from department store | |
fine tweezers | Fine Science Tools | 11254-20 | |
forceps | Fine Science Tools | 91106-12 | |
glass capillary pipettes, 1 mm OD, 0.58 mm ID | World Precision Instruments | 1B100F-3 | |
High Speed Rotary Micromotor Kit | Foredom Electric Co. | K.1070 – K.107018 | |
Ideal Micro Drill Burr Set Of 5 | Cell Point Scientific | 60-1000 | |
Impact One Stereotaxic Impactor for CCI | Leica Biosystems | 39463920 | |
isoflurane | Baxter | NDC-10019-360-60 | |
lab bench timers | Fisher Scientific | 14-649-17 | |
Micropipette puller | MicroData Instruments, Inc. | PMP-102 | Any puller will suffice |
Microscope cover slips | Fisherbrand | 12-545-E | |
Microscope slide mounting medium | Product of choice | ||
mirror | Purchase from department store | ||
mouse anti-human nuclear antigen antibody | Millipore | MAB1281 | |
Mouse on Mouse blocking kit | Vector Laboratories | BMK-2202 | |
needle holder hemostat | Fine Science Tools | 12002-12 | |
ophthalmic ointment | Falcon Pharmaceuticals | NDC-61314-631-36 | |
ophthalmic spring scissors | Fine Science Tools | 15018-10 | |
plastic box | Purchase from department store | ||
plastic cylinder | Purchase from department store | ||
QSI motorized syringe pump | Stoelting | 53311 | |
Removable needle compression fitting | Hamilton | 55750-01 | |
small rodent stereotaxic frame | Stoelting | 51925 | |
small scissors | Fine Science Tools | 14060-09 | |
StemPro Accutase | Invitrogen (ThermoFisher) | A1110501 | |
Sterile alcohol prep pads | Fisherbrand | 06-669-62 | |
sterile cotton swabs/Kendall Q-tips | Tyco Healthcare | 540500 | |
Sterile saline | Hospira | NDC-0409-1966-07 | |
Stopwatches (2) | Fisher Scientific | 06-662-56 | |
Superfrost Plus Gold microscope slides | Fisherbrand | 15-188-48 | |
sutures – 5.0 silk with curved needle | Oasis | MV-682 |