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.

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 (EL&#196;INKOELAUTAKUNTA-ELLA). Adult mice of 1-3 months age, weight 24-38 g, were kept in individual cages in the certified University&#8217;s animal facility and provided with food and water ad libitum.
1. Brain Injury Imaging Through a Cranial Window
<ol>
	<li>Anesthetizing animals and preparing the operation field
	<ol>
		<li>Anesthetize mice by peritoneal injection of a mixture of ketamine (Ketalar, 80 mg/kg) and xylazine (Rompun, 10 mg/kg) dissolved in filter-sterilized phosphate buffer saline (PBS), pH 7.4. Check the mouse&#8217;s reflexes regularly (tail and toe pinch probe) to verify the depth of anesthesia. Increase the dose when appropriate, to maintain proper anesthesia but avoid overdose.</li>
		<li>Maintain the animal at 37.0 &#176;C using a heating pad during surgical operation, imaging session and first hour after recovery from anesthesia.</li>
		<li>To protect the mouse&#8217;s eyes from drying out, apply eye lubricant.</li>
		<li>Administer dexamethasone (Rapidexon vet, 2 mg/kg) by subcutaneous injection to reduce inflammation and cerebral edema.</li>
		<li>Administer an analgesic (for instance, Ketoprophene intraperitoneally) up to 30 min prior to or immediately after the surgery. If the animal exhibits any pain symptoms, such as reluctance to move, eat or drink, or weight loss, salivation, piloerection, or abnormal respiratory sounds after surgery, repeat the injection of analgesic.</li>
		<li>Shave the mouse&#8217; head with a shaving machine (see Materials and Equipment). Avoid damaging the whiskers.</li>
		<li>Treat the skin on the mouse&#8217;s head with 70% ethanol solution to clean the shaved area.</li>
		<li>Using surgical scissors (see Materials and Equipment) and forceps (see Materials and Equipment), cut the skin from the middle line between ears to the forehead.</li>
		<li>Gently scrape away the connective tissue attached to the skull with a blunt microsurgical blade.</li>
		<li>Slide skin borders sideways and pull ear bars slightly into aural orifices of the skull for head and skin fixation.</li>
		<li>Clean the skull with sterile PBS and treat the surgery site on the mouse&#8217;s head with 0.5% chlorhexidine digluconate, then dry the skull surface using a combination of sterile cotton swabs and compressed air.</li>
	</ol>
	</li>
	<li>Infliction of the prick injury
	<ol>
		<li>Position a small animal stereotaxic instrument with an animal holder (see Materials and Equipment).</li>
		<li>Adjust the position of the binocular microscope next to the stereotaxic instrument to focus on the surface of the animal&#8217;s skull.</li>
		<li>Using a binocular microscope, locate the bregma point on the skull.</li>
		<li>Identify the region of interest using the stereotaxic coordinates. 
		NOTE: Avoid those areas of interest that are directly located over superficial cortical vessels. Destruction of these can strongly affect the trauma progression.</li>
		<li>Position the 30 G needle above the chosen region and mark the target site by scratching the skull surface.</li>
		<li>Drill the skull with all possible precautions to avoid unnecessary widening of the affected bone surface area. Use a high speed surgical drill (see Materials and Equipment) under the microscope. Stop drilling if liquid appears in the drilled area.</li>
		<li>Touch the bottom of the drilled well with the needle.</li>
		<li>Dip the needle smoothly into the brain (insertion rate 5-10 mm/min), to the depth of 0.5-2.0 mm according to the coordinates of the prick application site.</li>
		<li>Remove the needle immediately after reaching the desired depth (retraction rate 5-10 mm/min) and wipe the blood appearing after the prick with a small tampon (see Materials and equipment).</li>
		<li>Perform microinjection of 100 &#181;M Sulforhodamine 101 or another compound of interest into the lesion site using a microsyringe pump (WPI) and 10 &#181;l Hamilton syringe assemble with glass pipette. 
		NOTE: During preparation of the glass pipette from borosilicate glass capillary, sharpen the tip to a diameter of 10-20 &#181;m.</li>
		<li>Infuse 250-1,500 nl of the injection solution at the rate of 2-5 nl/sec. 
		NOTE: Leave the pipette after the end of infusion for at least 5 min in brain parenchyma, then remove it slowly and gently to prevent solution outflow.</li>
	</ol>
	</li>
	<li>Craniotomy for chronic cranial window
	<ol>
		<li>Drill the skull gently and carefully to make a circle window around the injury site. Use window diameter of 3-3.5 mm.</li>
		<li>Apply a drop of cortex buffer (125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl2 and 2 mM MgSO4 in distilled H2O) to cover the window.</li>
		<li>Position a round glass coverslip (#1.5 thickness) on the cranial window.</li>
		<li>Remove the excess of cortex buffer around the coverslip.</li>
		<li>Seal the edges of the coverslip with polyacrylic glue (see Materials and Equipment). 
		NOTE: Ensure that the glue is not applied to the upper surface of the coverslip. If the cover glass is contaminated with glue, wait until the glass is stably glued to the skull and the glue on the glass surface gets dry, then carefully remove the contaminating glue with a microblade.</li>
		<li>Wait for 5 min to let the glue dry up.</li>
		<li>Using the ear bar height screws, adjust the head position so that the metal holder can be placed.</li>
		<li>Apply a small amount of polyacrylic glue on the ring of the steel holder (see Materials and Equipment).</li>
		<li>Glue the ring of the steel holder to the glass coverslip with the metal holder handle directed backwards.</li>
		<li>Wait for 5 min to let the glue dry up.</li>
		<li>Mix dental cement (see Materials and equipment) with polyacrylic glue in a 3.5 cm Petri dish (or analogue) to achieve viscous conditions.</li>
		<li>Seal the borders of the cranial window with the cement + glue mixture and cover the exposed surface of the skull with the same mixture up to the skin border.</li>
		<li>Wait for 15 min to let the cement + glue mixture dry up and then remove ear bars.</li>
		<li>Perform two-photon excitation microscopic (TPEM) imaging for the region of interest and a control area as it described in Protocol 3.</li>
		<li>After imaging, allow the animal to recover from anesthesia on a heating pad and keep it in an individual cage under observation until full recovery. Wet food can be given to facilitate chewing and hydration.</li>
	</ol>
	</li>
</ol>
2. Brain Injury Imaging Through the Thinned Skull
<ol>
	<li>Anaesthetizing animals and preparing the operation field
	<ol>
		<li>Anesthetize the mouse and prepare it for trauma induction as described in step 1.1.</li>
	</ol>
	</li>
	<li>Skull thinning and infliction of the prick injury
	<ol>
		<li>Position a small animal stereotaxic instrument with the animal holder (see Materials and equipment).</li>
		<li>Adjust the position of a binocular microscope next to the stereotaxic instrument to focus on the animal skull surface.</li>
		<li>Locate the bregma point on the skull using the binocular microscope, identify the brain area to be imaged based on stereotactic coordinates and mark it by scratching. 
		NOTE: Skull thinning position should not be located over or nearby cranial sutures, as the skull stability is compromised in those areas. Furthermore, large cortical or meningeal vessels and meninges located below the skull sutures are likely to cause imaging artifacts.</li>
		<li>Place the metal holder coated with glue over the area of interest on the animal&#39;s skull, apply light pressure and wait for 5 min until the metal holder is firmly glued to the skull.</li>
		<li>Mix dental cement (see Materials and equipment) with polyacrylic glue in a 3.5 cm Petri dish (or analogue) to achieve viscous conditions.</li>
		<li>Seal the borders of the metal holder with the cement + glue mixture and cover the exposed surface of the skull with the same mixture up to the skin border.</li>
		<li>Wait for 15 min to let the cement and&#160;glue mixture dry up.</li>
		<li>Rinse the thinned skull region and the surrounding parts of the metal holder several times with PBS, so that the remnants of nonpolymerized glue are washed away. 
		NOTE: It is crucial to ensure stable attachment of skull to the holder. This enables the preparation stability during imaging.</li>
		<li>Using the high magnification mode of the binocular microscope, remove upper layers of the skull bone with a high-speed micro-drill to create a thinned skull area of ~0.5&#8211;1.5 mm in diameter. Use compressed air during drilling to remove the bone debris. Perform the drilling intermittently during the thinning procedure in order to avoid friction-induced overheating. Cool the drill bit using the room&#160;temperature solution and periodically apply buffer to the thinned area to absorb heat. 
		NOTE: To avoid damaging the cortex, do not drill over large regions (&#62;1.5 mm) down to a thin layer (&#60;50 &#956;m). 
		NOTE: The rodent skull bone structure consists of two thin layers of compact bone separated by a thick layer of spongy bone. Tiny cavities of the spongy bone form concentric circles and canaliculi, which contain blood vessels. Use the microdrill to remove both the external compact bone layer and most of the spongy layer. Disruption of the blood vessels within the spongy bone may cause bleeding. Use hemostasis collagen sponge to stop this bleeding.</li>
		<li>Use second-harmonic generation (SHG) imaging to examine whether the majority of the spongy bone has been removed. Be careful to avoid excessive thinning, make sure that the remaining bone is thicker than 50 &#181;m at this stage of the preparation.</li>
		<li>Put a drop of warm buffer (35-37 &#176;C) on top of the thinned region. Use a microsurgical blade or Micro Finishing Bur to further remove bone layers and form a smooth area of ~700 &#181;m in diameter with bone thickness of ~20 &#181;m. During this step, it is helpful to perform repetitive SHG imaging and measure regularly the bone thickness.</li>
		<li>Perform TPEM imaging for the region of interest and a control area.</li>
		<li>Adjust the head position using the ear bar height screws in such a way that thinned region is positioned strictly horizontally. Position the needle above the thinned region and make sure that there are no large vessels beneath the targeted site.</li>
		<li>Make a lesion by dipping the needle into the brain, to the depth of 0.5-2.0 mm from thinned skull surface and according to the coordinates of the prick application site.</li>
		<li>Remove the needle and suppress hemorrhage appearing after the acute brain injury with hemostatic tampon (see Materials and equipment). Wait until blood clots form and vessel pulsation at the injury site stops.&#160;</li>
		<li>Perform microinjection of 100 &#181;M Sulforhodamine 101 or another compound of interest into the lesion site, as described above in&#160;section 1.2.10.</li>
		<li>Perform TPEM imaging to monitor the injury progression and recovery as described in Protocol 3.</li>
		<li>After imaging, allow the animal to regain consciousness from anesthesia on a heating pad. Do not leave the animal unattended and return it to its home cage only after full physical recovery.</li>
	</ol>
	</li>
</ol>
3. Imaging
<ol>
	<li>Place the animal under the microscope by attaching the metal holder to the custom built frame.</li>
	<li>For imaging, use e.g. the FV1000MPE two-photon microscope equipped with Mai Tai DeepSee laser and XLPLN 25X 1.05 NA water immersion objective optimized for in vivo two-photon imaging.</li>
	<li>Identify the lesion site under the microscope using wide-field fluorescence mode. Use long pass filters to visualize brain vasculature and select the control area according to the observed pattern of blood vessels.</li>
	<li>To image the second harmonic generation (SHG) signal, tune the femtosecond laser to the 800 nm wavelength and collect the emitted light using the 380-410 nm bypass filter. For fluorescence imaging, use the band pass filter (515-560 nm) to collect emitted light, and the following wavelengths are used to excite the fluorescence: GFP-&#160;860 nm, YFP-&#160;950 nm.</li>
	<li>Use Fluoview software for image acquisition.</li>
	<li>Store coordinates of every ROI for subsequent repetitive imaging. Image the same ROIs over time, and adjust coordinates every time to maximize the image overlap.</li>
	<li>Analyze images with the appropriate software (e.g.&#160;ImageJ). Reconstructions presented here were done using Imaris software.</li>
</ol>

<ol>
	<li>Lighthall, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cortical+impact:+A+new+experimental+brain+injury+model.">Controlled cortical impact: A new experimental brain injury model.</a> J. Neurotrauma. 5 (1), 1-15 (1988).</li><li>Lindgren, S., Rinder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+in+head+injury.">Experimental studies in head injury.</a> Biophysik. 2 (5), 320-329 (1965).</li><li>Shreiber, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+cerebral+contusion:+histopathological+and+immunohistochemical+evaluation+of+dynamic+cortical+deformation.">Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation.</a> J. Neuropathol. Exp. Neurol. 58 (2), 153-164 (1999).</li><li>Feeney, D. M., Boyeson, M. G., Linn, R. T., Murray, H. M., Dail, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+to+cortical+injury:+I.+Methodology+and+local+effects+of+contusions+in+the+rat.">Responses to cortical injury: I. Methodology and local effects of contusions in the rat.</a> Brain Res. 211 (1), 67-77 (1981).</li><li>Bardehle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+astrocyte+responses+to+acute+injury+reveals+selective+juxtavascular+proliferation.">Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation.</a> Nat. Neurosci. 16 (5), 580-586 (2013).</li><li>Sword, J., Masuda, T., Croom, D., Kirov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+neuronal+and+astroglial+disruption+in+the+peri-contusional+cortex+of+mice+revealed+by+in+vivo+two-photon+imaging.">Evolution of neuronal and astroglial disruption in the peri-contusional cortex of mice revealed by in vivo two-photon imaging.</a> Brain. 136 (5), 1446-1461 (2013).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Nolte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP+promoter-controlled+EGFP-expressing+transgenic+mice:+a+tool+to+visualize+astrocytes+and+astrogliosis+in+living+brain+tissue.">GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue.</a> Glia. 33 (1), 72-86 (2001).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20 (11), 4106-4114 (2000).</li><li>Nimmerjahn, A., Kirchhoff, F., Kerr, J. N. D., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulforhodamine+101+as+a+specific+marker+of+astroglia+in+the+neocortex+in+vivo.">Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo.</a> Nat. Methods. 1 (1), 31-37 (2004).</li><li>Carr&#233;, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+aspects+of+an+impact+acceleration+traumatic+brain+injury+rat+model+with+potential+suitability+for+both+microdialysis+and+PtiO2+monitoring.">Technical aspects of an impact acceleration traumatic brain injury rat model with potential suitability for both microdialysis and PtiO2 monitoring.</a> J. Neurosci. Methods. 140, 23-28 (2004).</li><li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat. Protoc. 4 (8), 1128-1144 (2009).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat. Protoc. 5, 201-208 (2010).</li><li>Cianchetti, F. A., Kim, D. H., Dimiduk, S., Nishimura, N., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-evoked+calcium+transients+in+somatosensory+cortex+are+temporarily+inhibited+by+a+nearby+microhemorrhage.">Stimulus-evoked calcium transients in somatosensory cortex are temporarily inhibited by a nearby microhemorrhage.</a> PloS one. 8 (5), (2013).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J. Vis. Exp. 61, (2012).</li></ol>

The authors have nothing to disclose.

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&#8211;brain barrier. To study primary and secondary pathogenesis, as well as recovery after trauma, this injury model was combined with longitudinal...

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.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2A-sa dumb Tweezers, 115mm</td>
			<td style="width:71px;">XYtronic</td>
			<td style="width:119px;">XY-2A-SA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G &#189;&#8217;&#8217; needle</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">REF 304000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Animal trimmer, shaving machine</td>
			<td style="width:71px;">Aesculap</td>
			<td style="width:119px;">Isis GT420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binocular Microscope</td>
			<td style="width:71px;">Zeiss&#160;</td>
			<td style="width:119px;">Stemi 2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Biological Temperature Controller with stainless steel heating pad</td>
			<td style="width:71px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blunt microsurgical blade</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">REF 374769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Borosilicate tube with filament</td>
			<td style="width:71px;">Sutter Instruments</td>
			<td style="width:119px;">BF120-69-10</td>
			<td style="width:167px;">For glass pipette production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofene</td>
			<td style="width:71px;">Pfizer</td>
			<td style="width:119px;">Rimadyl vet</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chlorhexidine digluconate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C9394</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Dental cement</td>
			<td style="width:71px;">DrguDent, Dentsply</td>
			<td style="width:119px;">REF 640 200 271</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Dexamethasone</td>
			<td style="width:71px;">FaunaPharma</td>
			<td style="width:119px;">Rapidexon vet</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Disposable drills</td>
			<td style="width:71px;">Meisinger</td>
			<td style="width:119px;">HP 310&#160;104&#160;001&#160;001&#160;008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dulbeco&#8217;s PBS 10X</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D1408</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dumont #5 forceps, 110 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">91150-20</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Ealing microelectrode puller</td>
			<td style="width:71px;">Ealing</td>
			<td style="width:119px;">50-2013</td>
			<td style="width:167px;">Vertical puller for glass pipette production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eyes-ointment</td>
			<td style="width:71px;">Novartis</td>
			<td style="width:119px;">Viscotears</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foredom drill control</td>
			<td style="width:71px;">Foredom&#160;</td>
			<td style="width:119px;">FM3545</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foredom micro motor handpiece</td>
			<td style="width:71px;">Foredom</td>
			<td style="width:119px;">MH-145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Gas anesthesia platform for mice</td>
			<td style="width:71px;">Stoelting</td>
			<td align="right" style="width:119px;">50264</td>
			<td style="width:167px;">Assembled on stereotaxic instrument</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Hemostasis Collagen Sponge</td>
			<td style="width:71px;">Avitene, Ultrafoam BARD</td>
			<td style="width:119px;">Ref 1050050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaris</td>
			<td style="width:71px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:71px;">Intervet</td>
			<td style="width:119px;">Ketaminol vet</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Mai Tai DeepSee laser</td>
			<td style="width:71px;">Spectra-Physics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal holder</td>
			<td style="width:71px;">Neurotar</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro dressing forceps, 105 mm</td>
			<td style="width:71px;">Aesculap</td>
			<td style="width:119px;">BD302R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Microfil</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">MF34G-5</td>
			<td style="width:167px;">Micro syringe filling capillaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M8410</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Multiphoton Laser Scanning Microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">FV1000MPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NanoFil Syringe 10 microliter</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">NANOFIL</td>
			<td style="width:167px;">Hamilton syringe</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Nonwoven swabs 5&#215;5</td>
			<td style="width:71px;">Molnlycke Health Care</td>
			<td style="width:119px;">Mesoft</td>
			<td style="width:167px;">Surgical tampons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylic glue</td>
			<td style="width:71px;">Henkel</td>
			<td style="width:119px;">Loctite 401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Round glass coverslip&#160;</td>
			<td rowspan="2" style="width:71px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 thickness&#160;</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Small animal stereotaxic instrument</td>
			<td style="width:71px;">David Kopf Instruments</td>
			<td align="right" style="width:119px;">900</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Stoelting mouse and neonatal rat adaptor</td>
			<td style="width:71px;">Stoelting</td>
			<td align="right" style="width:119px;">51625</td>
			<td style="width:167px;">Assembled on stereotaxic instrument.</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Student iris scissors, straight 11.5 cm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">91460-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Sulforhodamine 101</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">S-359</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">UMP3-1</td>
			<td style="width:167px;">Microinjector and controller</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Xylazine</td>
			<td style="width:71px;">Bayer Health Care</td>
			<td style="width:119px;">Rompun vet</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

acute brain trauma in mice followed by longitudinal two-photon imaging

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.

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 &#956;m below the pial surface (Figure 1B), whereas skull thinning tends to impose a ...

Watch this Scientific Journal Video about Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging at JoVE.com

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

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 ...

acute-brain-trauma-in-mice-followed-by-longitudinal-two-photon-imaging

Research

JoVE Journal

Medicine

12.3K Views.  University of Helsinki. 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.

Video: Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...