Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.
Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.
Acute brain injury is a significant public health problem with high incidence of injury in motor vehicle crashes, falls or assaults, and high prevalence of subsequent chronic disability. Therapeutic approaches to the treatment of brain injury remain totally symptomatic, thus limiting the effectiveness of prehospital, surgical and critical care. This makes the social and economic impact of brain injury particularly severe. For a variety of reasons, most of the clinical trials failed to demonstrate improvement in recovery after brain injury using novel therapeutic approaches.
Animal models are crucial for developing new therapeutic strategies towards a stage where drug efficacy can be predicted in patients with brain injuries. At present, several well established animal models of head trauma exist, including controlled cortical impact1, fluid percussion injury2, dynamic cortical deformation3, weight-drop4, and photo injury5. A number of experimental models have been used to study certain morphological, molecular and behavioral aspects of head trauma-associated pathology. However, no single animal model is entirely successful in validating new therapeutic strategies. Development of reliable, reproducible and controlled animal models of brain injury is necessary to assess the complex pathological processes.
The novel combination of the latest microscopic imaging technologies and genetically-encoded fluorescent reporters offers an unprecedented opportunity to investigate all phases of brain injury, which include primary injury, spreading of the primary injury, secondary injury, and regeneration. In particular, in vivo two-photon microscopy is a unique nonlinear optical technology that allows real-time visualization of cellular and even subcellular structures in deep cortical layers of rodent brain. Several types of cells and organelles can be imaged simultaneously by combining different fluorescent markers. Using this powerful tool, we can visualize dynamic morphological and functional changes in living brain under posttraumatic conditions. The advantages of in vivo two-photon microscopy in studying brain injury were recently demonstrated by Kirov and colleagues6. Using a mild focal cortical contusion model, these authors showed that acute dendritic injury in the pericontusional cortex is gated by the decline in the local blood flow. Moreover, they demonstrated that the metabolically compromised cortex around the contusion site is further damaged by the spreading depolarization. This secondary damage affects synaptic circuitry, making the consequences of traumatic brain injury more severe.
Here, we propose the method of stereotaxic prick with a syringe needle, which could be combined with simultaneous topical drug application, as an advanced model for local brain injury and as a tool to study pathophysiological consequences of acute trauma in mammalian brain in vivo.
All the procedures presented here were performed according to local guidance for animal care (The Finnish Act on Animal Experimentation 62/2006). Animal license (ESAVI/2857/04.10.03/2012) was obtained from local authority (ELÄINKOELAUTAKUNTA-ELLA). Adult mice of 1-3 months age, weight 24-38 g, were kept in individual cages in the certified University’s animal facility and provided with food and water ad libitum.
1. Brain Injury Imaging Through a Cranial Window
2. Brain Injury Imaging Through the Thinned Skull
3. Imaging
We have optimized two operation procedures: 1) chronic cranial window and 2) skull thinning, for posttraumatic brain imaging in transgenic mice. Schematic view of the experimental preparations is presented in Figure 1. Traumatic prick by steel needle of 0.3 mm OD (30 G) is applied to the drilled well (Figure 1A). A successful cranial window preparation allows imaging at depths up to 650 μm below the pial surface (Figure 1B), whereas skull thinning tends to impose a limit of approximately 300 μm (Figure 1C), as demonstrated in the 3D reconstruction of Thy1-YFP-H mouse cortical pyramidal neurons.
The prick trauma results in elimination of dendrites and destruction of capillary networks in a controlled volume of the brain cortex. During the first two days, the lesion area increased and the trauma induced dendrite blebbing and formation of dendritic retraction bulbs in the perilesion areas, as observed using in vivo multiphoton microscopy (Figure 2).
We performed skull thinning to image activation and migration of microglia in CX3CR1-EGFP mice immediately after injury (Figure 3A). The SHG imaging offers a valuable tool to delineate precisely the injury site (Figure 3B). Extracellular matrix molecules that produce SHG signals are greatly enriched in brain parenchyma at the prick trauma. First, fine microglial processes are retrieved, then microglial cells migrate to the border of the injury site (Figure 3A).
To estimate potential injuries induced by thin glass pipette insertion and delivery of dye, we perform in vivo two-photon microscopy experiments with Sulforhodamine 101 microinjection in Thy1-YFP-H mice without brain trauma. Representative images shown in Figure 4 demonstrate microinjection site 3 hr after injection. The trace of pipette insertion can be seen in brain meninges visualized by SHG (Figure 4A). Astrocytes are labelled with Sulphorhodamine 101 introduced by the injection (Figure 4B). Dendrites expressing YFP under Thy1 promoter do not demonstrate any morphological signs of injury like blebbing or retraction bulbs (Figure 4C).
Figure 1. The method of acute brain injury in combination with cranial window or thin skull preparations combined with in vivo two-photon microscopy. A. Traumatic prick by steel needle of 0.3mm OD (30G) applied to the drilled well. The needle is briefly immersed into the brain 0.5-2 mm deep from the bottom of the well. B,C. 3D reconstruction of Thy1-YFP-H mouse cortical pyramidal neurons in yellow and schematic view of experimental preparations. The second harmonic generation (SHG) signal from thinned skull is shown in grey (C). D. Bright field view of the superficial blood vessels through the glass window immediately after the acute brain injury. E. Thinned skull before the injury application. The chosen region of interest (white frame) and the prick application site (red circle). A different area (red frame) of the thinned region should be imaged to monitor possible surgery-induced artifacts. Please click here to view a larger version of this figure.
Figure 2. Example of longitudinal multiphoton imaging of brain trauma development through the thinned skull. A. Top view of the injury site surrounded by YFP-labelled dendrites of cortical neurons 20 min after trauma infliction, as imaged through the thinned skull preparation. B. Magnified view of the area outlined in panel A. C. The same area of the brain as in B, reimaged 5 days after trauma. Dendrite blebbing is shown with white arrows, dendritic retraction bulbs – with red arrows. Please click here to view a larger version of this figure.
Figure 3. Examples of monitoring inflammation and glia activation during development of brain trauma using different markers suitable for in vivo multiphoton imaging e.g. fluorescent proteins, dyes, and second harmonic generation signal. The images were acquired 3 hr after the brain injury. A. GFP-expressing microglia (green) activation and migration after acute brain trauma imaged through cranial window in CX3CR1-EGFP mice; second harmonic generation (SHG) is shown in grey. B. GFP-expressing (green) and Sulforodamine 101-labeled (red) astrocytes in GFAP-EGFP mice around the injury site, which is outlined by strong second harmonic generation signal (grey) from extracellular matrix molecules. Arrows indicate examples of microglial cells (A) and astrocytes (B) after injury. The injury site border is identified with the SHG signal and depicted by dashed line. Please click here to view a larger version of this figure.
Figure 4. Examination of tissue impact made by solution injection via a glass micropipette. A. A trace (shown with dashed line) in SHG visualizing brain meninges 3 hr after injection. B. Astrocytes labelled with sulphorhodamine introduced by the injection. C. Dendrites expressing YFP under Thy1 promoter. D. Combined image of A (SHG – grey), B (astrocytes – red), C (neuronal dendrites – yellow). Please click here to view a larger version of this figure.
Brain trauma is an abrupt, unpredictable event. Here, we describe the animal model that reproduces a spectrum of pathological changes observed in human patients after brain injury such as neurodegeneration, elimination of dendrites, brain edema, glial scar, hemorrhages in the cerebral cortex coupled with focal subarachnoid hemorrhaging and increased permeability of the blood–brain barrier. To study primary and secondary pathogenesis, as well as recovery after trauma, this injury model was combined with longitudinal in vivo visualization of fine neuronal and glial structures. Transgenic mouse lines were used that express fluorescent proteins in neurons (Thy1-YFP-H)7, astrocytes (GFAP-EGFP)8, or microglia (CX3CR1-EGFP)9. Additionally, we used second harmonic generation (SHG) imaging and astrocyte loading with Sulfarhodamine 10110.
The model described here recapitulates penetrating type of brain injury. Therefore a limitation of the present model is that it does not provide information about mechanisms of closed head injury. Recently Sword and coauthors reported multiphoton imaging results on cell behavior in pericontusional cortex6. Taking into account that closed head injury is a more frequent medical case, the methodological approach reported by the authors is highly promising to complement traditional research methods in the field of impact brain trauma11.
We used both the chronic cranial window12 and the skull thinning13 preparations to study cell behavior under posttraumatic conditions in living brain. Both methods have certain advantages and limitations. Thus, the chronic cranial window provides better resolution, deeper optical penetration into the brain tissue and convenience for multiple imaging sessions. Conversely, skull thinning is less likely to induce inflammation at the imaging site, and, perhaps more importantly, it allows repetitive applications of drugs and dyes. A few examples of pharmacological agents to be applied in this type of experiments are given in Table 1.
Agent type | Injected agent | Areas of biology/pharmacy research |
Anti-inflammatory | Cyclosporin A | Traumatic brain injury, secondary damage, neurodegeneration |
Toxins | Tetrodotoxin | TBI – secondary damage, excytotoxicity |
Bicuculline | TBI – secondary damage, chloride homeostasis | |
Inhibitors of signaling pathways | PD98059 (MAPKK inhibitor) | signaling mechanisms of inflammation, secondary damage, posttraumatic cell death, neurodegeneration and regeneration |
SU6656 (Src family kinase inhibitor) | ||
Neurotrophic factors | Brain-derived neurotrophic factor, BDNF | TBI – neuronal survival after injury, neuroprotection in secondary posttraumatic brain damage |
Glial cell line-derived neurotrophic factor, GDNF | ||
Viruses | lentiviral vectors for protein expression | molecular mechanisms of posttraumatic neurodegeneration and regeneration, differential labelling of cell types in damaged brain for in vivo imaging |
adenoviral vectors for protein expression | ||
Ca2+ fluorescent indicators | Fluo-2,4 | signaling mechanisms of posttraumatic inflammation, cell death, cell migration, axonal/dendritic regeneration |
Oregon Green BAPTA |
Table 1. Pharmacological agents and dyes for cortical injection in prick injury model.
A wide range of biochemical events that are highly important under physiological and pathological conditions can be probed with chemical inhibitors, fluorescent indicators and mutant protein introduced via viral constructs. Advantages for choosing particular time window provided by the skull thinning preparation may be highly relevant for those studies.
In the present study, we used second harmonic generation signal to delineate the injury site. Alternatively, the injury site borders could be indicated by a weakly diffusing fluorescent dye, for instance a high molecular weight dextran fluorescent conjugate (2 million Dalton).
Recently, Schaffer and colleagues14 have used a chronic preparation with reopenable cranial window for repetitive delivery of fluorescent dyes to mouse cortex. It is likely that cranial window reopening may adversely affect the brain tissue transparency. Moreover, it is difficult to predict the time course of the reopening-induced inflammation, which may affect the connective tissue regrowth.
One major advantage of the thinned skull preparation is the possibility to deliver therapeutic compounds (and other materials requiring topical injection into brain tissue) multiple times during progression of the trauma, without complicating artifacts (e.g. without cranial window reopening).
If repetitive compound delivery directly to the injury site is desired, one should consider special means to prevent skull dry-out and bacterial infection. Thus, keeping the operated head region under sterile conditions and application of antibiotics (such as enrofloxacin) is recommended. To preserve the thinned skull region from drying, fill the metal holder with 1.5% agarose and cover it with round glass coverslip. In most cases this will allow maintaining the same transparency or thinned skull over the period of up to 10 days. Some additional should be taken if multiple imaging sessions in the thinned skull preparation are planned over a prolonged period of time. Immediately before each imaging session, gently remove the newly formed connective tissue from the thinned region using a microsurgical blade. Use the TPEM imaging of SHG to verify image quality and measure bone thickness. Tissue regrowth may compromise the penetration depth and cause image blurring accompanied by an increase in the background fluorescence. Refresh the skull thinning gently with a microsurgical blade to regain high imaging quality.
In those cases that do not require direct access to the injury site, we highly recommend covering the thinned region of the skull with a glass coverslip as described in the paper on polished and reinforced thinned skull preparation by Kleinfeld and colleagues15. This could be done by using the following optional procedure. Place a small drop of agarose (1.5%) on the thinned skull region, wait until it becomes a gel, remove all unnecessary agarose, i.e. leave it only on the injury site. Agarose should protect the injury site from external effects. Dry the thinned skull region using compressed air. Drop a small amount of polyacrylic glue on a small piece of #0 coverglass and place it on the thinned skull region. The polyacrylic glue prevents bone and connective tissue regrowth, thus keeping the thinned skull under the window preserved.
A combination of these procedures allows studying acute and chronic posttraumatic processes, delivering drugs topically or systemically and directly monitoring the treatment effects. Use of dual or triple transgenic mouse lines can also be beneficial, particularly for simultaneous imaging of multiple posttraumatic processes, such as glial scar formation and neuronal branch regrowth. We expect our models of acute brain injury studied with intravital two-photon microscopy to prove fruitful for drug candidate testing.
The authors have nothing to disclose.
We are deeply thankful to Dr. Frank Kirchhoff for providing GFAP-EGFP and CX3CR1-EGFP mouse strains. The work was supported by grants from the Centre of International Mobility of Finland, Tekes, Finnish Graduate School of Neuroscience (FGSN) and the Academy of Finland.
2A-sa dumb Tweezers, 115mm | XYtronic | XY-2A-SA | |
30G ½’’ needle | BD | REF 304000 | |
Animal trimmer, shaving machine | Aesculap | Isis GT420 | |
Binocular Microscope | Zeiss | Stemi 2000 | |
Biological Temperature Controller with stainless steel heating pad | Supertech | TMP-5b | |
Blunt microsurgical blade | BD | REF 374769 | |
Borosilicate tube with filament | Sutter Instruments | BF120-69-10 | For glass pipette production |
Carprofene | Pfizer | Rimadyl vet | |
Chlorhexidine digluconate | Sigma | C9394 | |
Dental cement | DrguDent, Dentsply | REF 640 200 271 | |
Dexamethasone | FaunaPharma | Rapidexon vet | |
Disposable drills | Meisinger | HP 310 104 001 001 008 | |
Dulbeco’s PBS 10X | Sigma | D1408 | |
Dumont #5 forceps, 110 mm | FST | 91150-20 | |
Ealing microelectrode puller | Ealing | 50-2013 | Vertical puller for glass pipette production |
Eyes-ointment | Novartis | Viscotears | |
Foredom drill control | Foredom | FM3545 | |
Foredom micro motor handpiece | Foredom | MH-145 | |
Gas anesthesia platform for mice | Stoelting | 50264 | Assembled on stereotaxic instrument |
Hemostasis Collagen Sponge | Avitene, Ultrafoam BARD | Ref 1050050 | |
Imaris | Bitplane | ||
Ketamine | Intervet | Ketaminol vet | |
Mai Tai DeepSee laser | Spectra-Physics | ||
Metal holder | Neurotar | ||
Micro dressing forceps, 105 mm | Aesculap | BD302R | |
Microfil | WPI | MF34G-5 | Micro syringe filling capillaries |
Mineral oil | Sigma | M8410 | |
Multiphoton Laser Scanning Microscope | Olympus | FV1000MPE | |
NanoFil Syringe 10 microliter | WPI | NANOFIL | Hamilton syringe |
Nonwoven swabs 5×5 | Molnlycke Health Care | Mesoft | Surgical tampons |
polyacrylic glue | Henkel | Loctite 401 | |
Round glass coverslip | Electron Microscopy Sciences | ||
1.5 thickness | |||
Small animal stereotaxic instrument | David Kopf Instruments | 900 | |
Stoelting mouse and neonatal rat adaptor | Stoelting | 51625 | Assembled on stereotaxic instrument. |
Student iris scissors, straight 11.5 cm | FST | 91460-11 | |
Sulforhodamine 101 | Molecular Probes | S-359 | |
UMP3 microsyringe pump and Micro 4 microsyringe pump controller | WPI | UMP3-1 | Microinjector and controller |
Xylazine | Bayer Health Care | Rompun vet |