This work was supported by the Dr. Werner Jackst&#228;dt Foundation (to Eduardo Sanchez-Mendoza), the German Academic Exchange Service (DAAD; to Jeismar Carballo), the German Research Council (HE3173/2-1, HE3173/2-2, and HE3173/3-1; to Dirk M. Hermann), Heinz Nixdorf Foundation (to Dirk M. Hermann).

Animal experiments were performed with government approval&#160;(G1361/13, AZ84-02.04.2013.A192 and G1362/13, AZ84-02.04.2013.A194; Bezirksregierung D&#252;sseldorf) based on NIH Guidelines for the Care and Use of Laboratory Animals.
1. Preparation of Miniosmotic Pumps
<ol>
	<li>In a sterile environment (i.e., cell culture hood), obtain the pump, catheter, flow moderator, brain infusion cannula and the spacer discs to be used (Figure 1).</li>
	<li>For delivery of drugs directly into the ventricles use one spacer disc so that only the tip of the cannula needle is in contact with the ventricle. Hold the cannula upside down with a forceps. Add two drops of cyanoacrylate adhesive (C.A.) and introduce one spacer disc.</li>
	<li>Leave the cannula on a flat surface facing upwards so that the glue between the spacer disc and the cannula dries.</li>
	<li>Cut the catheter in sections of about 2.5 cm.</li>
	<li>For each pump, prepare a minimal of 300-350 &#181;l of the solution of interest as it will be needed to completely fill the pump thus extruding all air inside of it to prevent the formation of bubbles within the catheter. Bubbles will impede the flow of the solution into the brain.</li>
	<li>Carefully connect the catheter to the flow moderator.</li>
	<li>Using a 2 ml syringe connected to the needle provided by the brain infusion kit, fill the pump until a little amount of solution escapes the pump, thus preventing air bubbles inside it.</li>
	<li>Carefully fill the flow moderator and catheter paying attention to prevent any bubbles remaining inside the catheter.</li>
	<li>Introduce the flow moderator inside the pump.</li>
	<li>Once filled, carefully connect the cannula to the end of the catheter. If it is observed that a bubble is formed remove the cannula and carefully refill the catheter with vehicle or drug solution and then reintroduce the cannula.</li>
	<li>Place the pump in a container with sterile saline solution and leave it at 37 &#176;C O/N.</li>
	<li>Before implantation recheck for bubble formation. If necessary (i.e., bubbles are observed) remove the cannula and refill the tube. Reconnect the cannula. This should take only a few microliters of solution.</li>
</ol>
2. Implantation of Miniosmotic Pumps
Note: For these experiments animals were anesthetized by 1% isoflurane (30% O2, 70% N2O). However if this is not available, the use of intraperitoneal injections of anesthesia is also possible11.
<ol>
	<li>Locate the animal in the stereotactic device under anesthesia and cover the eyes with a protective ointment to prevent dryness while under anesthesia. Confirm anesthesia by observing a lack of response in the hindpaw when pressed with the fingers or with a forceps. Do not proceed until the animal is completely asleep.</li>
	<li>Cut the fur over the head either with scissors or a shaving machine. Cut as much as possible without damaging the skin.</li>
	<li>Clean the skin with 70% ethanol and disinfectant with antibacterial and fungicide properties.</li>
	<li>With a scalpel open a 1 cm incision slightly to the right of the midline and expose the skull.</li>
	<li>Clean the skull with a cotton swab. Soak it with 70% ethanol and pass it over the skull. This will induce the skull to dry.</li>
	<li>If the skull has light bleeding, use a cauterizer to eliminate any bleeding points. Blood prevents the C.A. from drying properly over the bone once the cannula is implanted.</li>
	<li>Use the stereotactic device to make a mark on the point of the skull where the cannula will be implanted. For ventricular infusions on the left hemisphere coordinates are -0.2 mm caudal, 0.9 mm lateral to bregma (Figure 2A).</li>
	<li>Gently drill over the skull making a 45&#176; angle; this prevents the drill from accidentally going into the brain. Repeatedly drill for a few seconds and then check how deep the hole is. Stop drilling once the skull has been thinned but still not fully penetrated.</li>
	<li>Break the meninges with the tip of a sterile needle until full access to the brain is achieved. Do so gently so as to not damage the brain.</li>
	<li>Clean the skull with 70% ethanol using a cotton swab.</li>
	<li>Introduce a straight forceps under the skin of the animal in an antero-caudal direction. Use the forceps to open the space under the skin in the back of the animal where the pump will be implanted. Introduce the pump into the back of the animal leaving the catheter and cannula outside (Figure 2B). The pump will remain in this position until it is removed and does not need any kind of fixation.</li>
	<li>Carefully place four little drops of glue next to the needle in the cannula.</li>
	<li>Carefully introduce the needle through the skull without moving it sideways. Hold it in position for 15-30 sec until the cannula is completely attached (Figure 2C.1). Once in place, the cannula will reach 2.5 mm in the dorso ventral direction if one spacer disc has been used. If properly attached, the cannula will remain attached to the bone until the end of the experiment.</li>
	<li>Place one finger over the removable tab and then use the other hand to cut it off by its neck with a set of scissors (Figure 2C.2).</li>
	<li>Close the wound on the skin with a 5-0 suture and add a few drops of povidone-iodine solution (PVP-I) on top of the wound to prevent infection (Figure 2D).</li>
	<li>Move the animal into a new cage. Do not place operated animals with animals that have still not been operated. Keep animals that have been implanted with pumps alone in their cages during the time of drug administration.</li>
	<li>Do not leave mice unattended until they are awake and have regained sternal recumbency.</li>
</ol>
3. Pump Removal
Note: Usually the experiment will end at the end of the delivery time allowed by the pump, however it is possible to remove the pump in order to do secondary experiments as a follow up to the drug delivery. In order to do tract tracer injections it is thus necessary to remove the pump.
<ol>
	<li>Place the animal in the seterotactic device under anesthesia and cover the eyes with protective ointment to prevent dryness while under anesthesia. Confirm anesthesia by observing a lack of response in the hindpaw when pressed with the fingers or with a forceps. Do not proceed until the animal is completely asleep.</li>
	<li>Carefully open the skin by cutting through the incision done on the day of the pump implantation.</li>
	<li>With a surgical clamp hold the cannula and pull it out. It should be easily removed from the skull. As gentle bleeding is expected, stop it by placing a cotton swab and wait for 1-2 min.</li>
	<li>Pull the pump out by the catheter. Help it come out by pushing it, creating pressure over the skin.</li>
	<li>Close the wound again with a 5-0 suture and add a few drops of PVP-I.</li>
	<li>Move the animal into a new cage. Do not put operated animals with animals that have still not been operated. Animals that have undergone surgery can be put together.</li>
	<li>Do not leave mice unattended until they are awake and have regained sternal recumbency.</li>
	<li>Allow the animal to recover for 10 days before proceeding to tract tracer injection.</li>
</ol>
4. Pressure Tract Tracer Injection at 45&#176; Angles on the Right Motor Cortex
<ol>
	<li>Place the animal on the stereotactic device under anesthesia and cover the eyes with a protective ointment to prevent dryness while under anesthesia. Confirm anesthesia by observing a lack of response in the hindpaw when pressed with the fingers or with a forceps. Do not proceed until the animal is completely asleep.</li>
	<li>Open the head wound.</li>
	<li>Clean the skull with 70% Ethanol using a cotton swab.</li>
	<li>Introduce a 5 &#181;l glass syringe (26S ga) on the vertical holder of the device. Make sure the lower end of the glass holder is precisely at the holding piece. The syringe must not be too far below it as then it will be impossible to place it at 45&#176; from the vertical position and perform the injection.</li>
	<li>Orient the syringe directly over bregma and then locate the desired coordinates. For an injection on the motor cortex coordinates are: Point #1: +0.5 mm rostral and 2.5 mm lateral with respect to bregma. Point #2: +1.34 mm rostral, 2.5 mm lateral with respect to bregma.</li>
	<li>Once coordinates have been located, indicate their position over the skull with a marker with a thin tip. Make only one dot in the skull as excessive ink release might hide the correct coordinate.</li>
	<li>Carefully drill the skull holding the drill at 45&#176;. As in step 2.8, check the skull frequently so that it is not fully perforated in order to avoid damage to the brain caused by the drill. Use the tip of a syringe to ensure that all bone has been removed.</li>
	<li>Load 600 nl of tract tracer dilution into the syringe.</li>
	<li>Adjust the vertical position to 45&#176; towards the right side of the animal. Under the microscope, place the tip of the syringe&#8217;s needle right in front of the hole.</li>
	<li>Move it in the vertical direction by 1.5 mm. Let the needle steady in this position for 30 sec to 1 min before doing pressure injections.</li>
	<li>Inject 300 nl of tracer dilution in three steps of 100 nl separated from each other by 30 sec. After the last injection, leave the syringe steady for 30 sec to 1 min in order to avoid the tracer to flow out of the brain.</li>
	<li>Slowly pull out the syringe, relocate it over the second hole and repeat the same process with the remaining 300 nl that are inside the syringe.</li>
	<li>After the second injection, remove the syringe and proceed to close the wound with a 5-0 suture and add a few drops of PVP-I.</li>
	<li>Move the animal into a new cage. Do not put operated animals with animals that have still not been operated. Animals that have undergone surgery can be put together. Do not leave mice unattended until they are awake and have regained sternal recumbency.</li>
	<li>Allow the animal to recover for 10 days before sacrifice.</li>
</ol>
5. Tract Tracer Observation
<ol>
	<li>Anesthetize the animal fully per institutional guidelines. Confirm anesthesia by observing a lack of response in the hindpaw when pressed with the fingers or with a forceps. Do not proceed until the animal is completely asleep.</li>
	<li>Perfuse the animals with 4% paraformaldehyde in PBS (pH 7) under standard protocols6.</li>
	<li>Remove the brain and post fix it by O/N immersion in 4% paraformaldehyde.</li>
	<li>Cryoprotect brains in sucrose at 30% until the brains sink. Then remove the brains and freeze them by 10 sec immersion in liquid nitrogen. Keep brains at -80 &#176;C until sections are produced.</li>
	<li>Produce sections at 20 &#181;m for retrograde tract tracer analysis and at 40 &#181;m for anterograde tract tracer analysis by sectioning on a cryostate.</li>
</ol>
Note: FG labelled neurons can be observed as white cells under ultraviolet light excitation. BDA is detected by O/N incubation with an avidin-biotin-peroxidase complex and 3,3&#8217; diaminobenzidine with addition of nickel at 0.4% to enhance contrast of the fibers6,7.

<ol>
	<li>Doeppner, T. R., 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=MicroRNA-124+protects+against+focal+cerebral+ischemia+via+mechanisms+involving+Usp14-dependent+REST+degradation.">MicroRNA-124 protects against focal cerebral ischemia via mechanisms involving Usp14-dependent REST degradation.</a> Acta Neuropathol. 126, 251-265 (2013).</li><li>Hoyo-Becerra, 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=Rapid+Regulation+of+Depression-Associated+Genes+in+a+New+Mouse+Model+Mimicking+Interferon-alpha-Related+Depression+in+Hepatitis+C+Virus+Infection.">Rapid Regulation of Depression-Associated Genes in a New Mouse Model Mimicking Interferon-alpha-Related Depression in Hepatitis C Virus Infection.</a> Mol Neurobiol. , (2014).</li><li>Puntel, M., 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=Gene+transfer+into+rat+brain+using+adenoviral+vectors.">Gene transfer into rat brain using adenoviral vectors.</a> Curr Protoc Neurosci. Chapter 4, Unit 4.24 (2010).</li><li>Miao, J., 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=Overexpression+of+adiponectin+improves+neurobehavioral+outcomes+after+focal+cerebral+ischemia+in+aged+mice.">Overexpression of adiponectin improves neurobehavioral outcomes after focal cerebral ischemia in aged mice.</a> CNS Neurosci Ther. 19, 969-977 (2013).</li><li>Pellegrini, L., 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=Therapeutic+benefit+of+a+combined+strategy+using+erythropoietin+and+endothelial+progenitor+cells+after+transient+focal+cerebral+ischemia+in+rats.">Therapeutic benefit of a combined strategy using erythropoietin and endothelial progenitor cells after transient focal cerebral ischemia in rats.</a> Neurol Res. 35, 937-947 (2013).</li><li>Reitmeir, R., 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=Post-acute+delivery+of+erythropoietin+induces+stroke+recovery+by+promoting+perilesional+tissue+remodelling+and+contralesional+pyramidal+tract+plasticity.">Post-acute delivery of erythropoietin induces stroke recovery by promoting perilesional tissue remodelling and contralesional pyramidal tract plasticity.</a> Brain. 134, 84-99 (2011).</li><li>Reitmeir, R., 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=Vascular+endothelial+growth+factor+induces+contralesional+corticobulbar+plasticity+and+functional+neurological+recovery+in+the+ischemic+brain.">Vascular endothelial growth factor induces contralesional corticobulbar plasticity and functional neurological recovery in the ischemic brain.</a> Acta Neuropathol. 123, 273-284 (2012).</li><li>Hermann, D. M., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoting+brain+remodelling+and+plasticity+for+stroke+recovery:+therapeutic+promise+and+potential+pitfalls+of+clinical+translation.">Promoting brain remodelling and plasticity for stroke recovery: therapeutic promise and potential pitfalls of clinical translation.</a> Lancet Neurol. 11, 369-380 (2012).</li><li>Overman, J. J., 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=A+role+for+ephrin-A5+in+axonal+sprouting,+recovery,+and+activity-dependent+plasticity+after+stroke.">A role for ephrin-A5 in axonal sprouting, recovery, and activity-dependent plasticity after stroke.</a> Proc Natl Acad Sci U S A. 109, E2230-E2239 (2012).</li><li>Wolf, W. A., Martin, J. L., Kartje, G. L., Farrer, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Fibroblast+Growth+Factor-2+as+a+Mediator+of+Amphetamine-Enhanced+Motor+Improvement+following+Stroke.">Evidence for Fibroblast Growth Factor-2 as a Mediator of Amphetamine-Enhanced Motor Improvement following Stroke.</a> PLoS One. 9, e108031 (2014).</li><li>Arras, M., Autenried, P., Rettich, A., Spaeni, D., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intraperitoneal+injection+anesthesia+in+mice:+drugs,+dosages,+adverse+effects,+and+anesthesia+depth.">Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.</a> Comp Med. 51, 443-456 (2001).</li></ol>

Production and Open Access publication fees provided by DURECT Corporation, which produces the miniosmotic pumps used in this Article.

For many years, research on neurodegenerative conditions like ischemic stroke or traumatic brain injury has focused on development of neuroprotective therapies that aim to promote neuronal survival in the acute stroke phase. The vast majority of drug therapies that have been found to be effective in rodent models failed when translated to the clinic. Reasons for this therapeutic failure include but are not restricted to the lack of sustained drug effects resulting in persisting functional neurological recovery. It is thus important to develop strategies promoting brain remodeling in the longer run. Because the promotion of neuronal survival alone is not sufficient to allow successful stroke recovery, as suggested by the large number of unsuccessful neuroprotection trials, the stimulation of neuronal plasticity has recently obtained major interest in the field.
Means for drug delivery are intraperitoneal injection, tail intravascular injection, femoral injection, single stereotactic injection of vectors into the brain and continued constant delivery by miniosmotic pumps. The latter can include systemic delivery, if the pump does not have a cannula, or which can be organ-directed, as we have shown for delivery into the brain. With the exception of miniosmotic pumps and the use of viral vectors, all other strategies will induce fluctuating drug concentrations. For long term experiments it thus becomes necessary to submit the animal to the stress of receiving frequent injections. The BBB imposes an important impediment for the brain uptake of proteins or drugs from the blood, resulting in the need of huge protein or drug dosages in order to achieve therapeutic concentrations in the brain. For example Pellegrini et al. (2013) 5 delivered rhEpo by intraperitoneal injection at a dose equivalent to 75 IU/day for an animal of 30 g (750 IU/day for a 300 g rat). In comparison, the targeted delivery of rhEpo to the brain allowed us to use a much lower dose of only 10 IU/day in our study for successful stroke recovery, which enabled us to achieve recovery over a large time scale at a fixed rate of 0.25 &#181;l/hr.
In this work we have shown the method of implantation of minipumps with a cannula connected to the skull in order to deliver the plasticity-promoting protein rhEpo directly into the ventricle, thus circumventing the BBB. By this method, rhEpo promoted neurological recovery in a number of ways, including reduction of infarct size, reduction of glial scar formation and induction of angiogenesis. rhEpo also promoted neuronal survival and increased projections from the contralesional motor cortex towards the denervated red nucleus and facial nuclei. The sprouting of the fibers was revealed by injection of the anterograde tract tracer BDA into the motor cortex (Figures 4A and 5A). A functional correlate to the sprouting of the fibers is provided by the improvement of motor skills (Figure 5B). Additionally, we have shown that the same approach for tract tracer injection can be applied to unveil thalamo-cortical connections by injection of the retrograde tract tracer FG (Figure 6B).
In the preparation of the miniosmotic pump, it is critical to consider the target point and the use of spacers. We use one spacer to reduce the length of the needle by 0.5mm as in this way the very tip of the needle is in contact with the ventricle at the given coordinates (-0.2 mm caudal, 0.9 mm lateral, 2.5 mm dorso ventral, with respect to bregma). However if deeper structures are the target of the research, then no spacers will be needed. Likewise, if a more external delivery point is desired (i.e., the cortex), then more spacer discs will be necessary. The catheter must be long enough so that the pump is not too close to the head, as it will impede movements of the mouse, but also not too long as once implanted excessive length may cause the catheter to bend, thus increasing the risk of cannula removal by the natural movement of the mouse. A section of 2 cm of catheter gives very good results in terms of mobility and stability of the implant (Figures 1 and 2). Incubation of the pump at 37 &#176;C O/N allows the pump to immediately start pumping the drug into the brain at the moment of implantation.
In the miniosmotic pump implantation it is critical to assure that the skull is properly dried before implanting the cannula. Usually cleaning with 70% Ethanol will induce the bone to dry, but if continuous bleeding is found, touching the skull gently with a cauterizer will completely dry it. It is critical to assure that the introduction of the needle is as vertical and slow as possible. Once in position, and while the glue is drying, placing the finger on top of the cannula prevents it from moving sideways over the skull. Special care should be given to the wound and placement of the cannula. It is important that the incision is not performed exactly over the middle line of the skull but slightly to the right side. When closing the wound, if the incision was made at the middle line, the skin will be overstretched, thus increasing the risk of wound opening. Making the incision slightly to one side will allow the suture points to be away from the highest part of the cannula. As a consequence there will be less tension on the suture points and the wound will heal properly. Animals should be caged alone and checked every day, especially during the first 10-15 days after the implantation. In case of wound dehiscence, wounds have to be closed as soon as possible. If the cannula is removed or the animal presents an infection, the experiment has to be terminated. Re-implantation of the cannula is not recommended. It is very important for successful implantation to use adequate amounts of tissue adhesive (not too much!) as it degrades the bone and increases the risk of cannula removal. However using too little adhesive will also not hold the cannula attached to the bone. The miniosmotic pumps can carry drugs dissolved in a wide variety of substances, being the only limitation to this that the solvent is biocompatible. Additionally, given that the volume is small (200 &#181;l) one must determine whether the concentration required for the experiment is suitable and will not cause precipitation inside the pump.
Tract tracing with either anterograde or retrograde tracers is a very well established technique to study brain connectivity and plasticity. Care must be given to the use stereotactic frames when injecting to ensure accuracy on targeting the brain area one wishes to study (i.e., to prevent injection on the corpus callosum when injecting the cortex).
For all surgical interventions and in order to reduce pain and inflammation, animals should be treated with 0.1 mg/kg Buprenorphine before the intervention and Caprofen at 4 mg/kg once a day for three days after the intervention.
In conclusion, this approach provides a proper tool for studying effect of proteins or pharmacological compounds in the injured brain, representing a method that is well suited for studies on brain plasticity.

The delivery of proteins and pharmacological compounds into the brain are important strategies for studying mechanisms underlying brain diseases and evaluating candidate molecules for new treatments 1,2. In experimental neurosciences, the delivery of vectors such as plasmids or adenoviruses has become an important tool for studying long-term actions of proteins in the brain 3,4. Single injections of vectors present the advantage of a system which by itself will maintain highly stable levels of the therapeutic agent in the brain 4. However, for long term experiments with purified drugs systemic administration by intraperitoneal injection induces stress in mice or rats, and is not the best choice when a targeted brain response is needed, requiring also large doses of drug5. Miniosmotic pumps represent an ideal system for prolonged direct drug delivery into the brain by circumventing both low accessibility to the brain and also peaks of drug concentration, as the delivery of the drug happens directly into a targeted place in the brain and at a fixed flow rate determined by the pump model that is chosen2,6,7. Indeed, this system has allowed us to successfully study brain recovery after stroke by delivery of several drugs such as recombinant human erythropoietin (rhEpo) and vascular endothelial growth factor 6,7. 
Brain plasticity is essential for the rewiring of connections in response to brain injuries. Plasticity is a broad concept that ranges from the formation or elimination of synaptic contacts, growth of dendritic spines and also elongation or retraction of long distance connections8,9. The brain was previously believed to not be capable of reconstructing connections after a lesion. However many approaches have shown that if properly stimulated it can reestablish connectivity 6,7,10. One technique that is particularly useful to study this is the use of tract tracers. Anterograde tract tracers are compounds that can enter neurons at the soma and then distribute all along the axons until these reach their target structures. Two examples are cascade blue (CB) and biotinylated dextran amine (BDA). Conversely, retrograde tract tracers, such as cholera toxin B (CTB) or fluorogold (FG) enter the neuron through the axon terminal and then distribute back to the soma thus revealing the site of origin of neurons targeting the injection site.
Here, we present the methods that we use for implantation of miniosmotic pumps for direct delivery of proteins or drugs that have potential effects on neural plasticity as well as the injection of BDA and FG to unveil input and output connections to the motor cortex. BDA will also be used as an example of a tract tracer used to demonstrate increased plasticity of axons emerging from the co after stroke under rhEpo treatment.

<table><tbody><tr><td>Alzet miniosmotic pump. Model 2004.</td><td>Alzet</td><td>000298</td><td>Drug container</td></tr><tr><td>Brain infusion kit 3 1-3 mm</td><td>Alzet</td><td>0008851</td><td>Drug brain delivery system</td></tr><tr><td>Loctite&amp;nbsp; 454 Prism gel&amp;nbsp;</td><td>Loctite</td><td>45404</td><td>Cyanoacrylate adhesive for cannula adhesion to the skull</td></tr><tr><td>75N glass syringe&amp;nbsp;</td><td>Hamilton</td><td>87900/00</td><td>Injection of tract tracers</td></tr><tr><td>Biotin Dextran Amine (10,000 MW)</td><td>Molecular probes</td><td>N-7167</td><td>Anterograde tract tracer</td></tr><tr><td>Fluorogold</td><td>Fluorochrome, LLC.</td><td></td><td>Retrograde tract tracer</td></tr><tr><td>Quintessential Stereotaxic Injector (QSI)</td><td>Stoelting</td><td>53311</td><td>Stereotactic device for coordinate determination, pump implantation and tract tracer injection.</td></tr></tbody></table>

implantation of miniosmotic pumps and delivery of tract tracers to study brain reorganization in pathophysiological conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

We submitted animals to 30 min of middle cerebral artery occlusion by the intraluminal suture method inducing a lesion in the left striatum and then delivered rhEpo directly into the brain by means of miniosmotic pumps (Figure 1, Figure 3) during 30 days starting 3 days after stroke6. Figure 4 shows a schematic of the cortico spinal tract that was traced after CB and BDA injection and the area where tracers were injected. We showed an improvement of grip strength and motor performance (Figure 5) after 14 and 42 days of rhEpo delivery respectively. Delivery of BDA into the right motor cortex of animals that received a stroke on the left striatum, showed an increase in motor fibers crossing the mid line at the level of the red and facial nucleus (Figure 5), demonstrating successful staining of sprouting fibers as a consequence of pharmacological treatment with miniosmotic pumps. rhEpo treatment also increased neuronal survival, delayed diffuse astrocytosis, reduced glial scar formation and increased angiogenesis in the studied period6. By using this same technique for tract tracer injection we can successfully detect thalamic nuclei that are connected to the cortex by injection of the retrograde tract tracer FG (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/52932/52932fig1.jpg" /> 
Figure 1.&#160;Components of the miniosmotic pump used in this protocol. The spacer disc, cannula and removable tab, catheter, flow moderator and miniosmotic pump can be observed. The aspect of the fully assembled pump can be seen in Figure 2A. <a href="https://www.jove.com/files/ftp_upload/52932/52932fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52932/52932fig2.jpg" /> 
Figure 2.&#160;Summary of pump implantation key points.&#160;(A) The mouse is shown as placed on the stereotactic device with the fully constructed miniosmotic pump next to it. Arrow indicates the coordinates selected for implantation. (B) The pump has been introduced on the back of the animal and only the cannula remains on the exterior. The skull has already been drilled. (C) Aspect of the head after implantation. (C.1) The cannula is on position but the removable tab has not been cut. (C.2) The removable tab has been cut and stitching of the wound can now begin. (D) The asterisk shows a recently implanted animal as compared to an animal 30 days after implantation (#). When housed correctly, the wound should remain closed until the end of the procedure as shown on the image. <a href="https://www.jove.com/files/ftp_upload/52932/52932fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52932/52932fig3.jpg" /> 
Figure 3.&#160;Nissl staining indicating the site of implantation on the cortex. A small incision can be observed on part of the left cortex (arrow). The width of the penetrated area is approximately 50 &#956;m. The are no evident severe tissue alterations based on Nissl staining as compared to the corresponding contralateral area (*). R: Right hemisphere. L: Left hemisphere. Scale bar = 300 &#956;m. <a href="https://www.jove.com/files/ftp_upload/52932/52932fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52932/52932fig4.jpg" /> 
Figure 4.&#160;Tract tracer injection strategy as published before by Reitmeier et al.6,7.&#160;(A) Schematic indicating injection sites for the tract tracer BDA at the contralateral motor cortex whereas CB was injected in the motor cortex of the infarcted hemisphere. Fibers were followed to the red nucleus (not shown) and facial nucleus (see Figure 5). (B) The injection site of the anterograde tract tracer BDA next to the motor cortex is shown. Note the red arrow indicating the needle track whereas the black arrow shows a few cortical cells labelled with the BDA. Cx: Cortex. CC: Corpus callosum. V: Ventricle. FN: Facial nucleus. Scale bar in B = 200 &#181;m. Figure 4A is reproduced with kind permission6. <a href="https://www.jove.com/files/ftp_upload/52932/52932fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52932/52932fig5.jpg" /> 
Figure 5.&#160;Recovery of an infarcted brain after delivery of rhEpo.&#160;(A) BDA injected in the contralesional motor cortex is then detected in corticobulbar fibers at the level of the facial nucleus (Bregma -5.8 mm to -6.3 mm). Intersection lines on each hemisphere were drawn parallel to the midline and fibers crossing each line in direction to the ipsilesional and contralesional hemisphere were counted and expressed as percentage of total labelled fibers in the corticospinal tract. Erythropoietin increased fiber crossings in direction to the contralesional facial nucleus Data are means +- SD. Data were analysed by one-way ANOVA followed by least significant differences tests, &#167;P&#60;0.05 compared with vehicle-treated non-ischaemic mice. (B) Motor behavior showed an improvement of hand grip strength and coordination in the rota rod test. Data are mean values +- SD. Data were analysed by two-way repeated measures ANOVA, followed by one-way ANOVA/least significant differences tests for each time-point. &#167;P&#60;0.05 compared with pre-ischaemic baseline; *P&#60;0.05 compared with vehicle treated ischemic mice. Figures 5A and B are reproduced with kind permission6. <a href="https://www.jove.com/files/ftp_upload/52932/52932fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52932/52932fig6.jpg" /> 
Figure 6.&#160;Target structures reached after injection of fluorogold (FG).&#160;(A) Injection site of FG next to the motor cortex as indicated in Figure 1. Note the red arrow indicating the needle track and the white arrow indicating a few labelled cells in the cortex. (B) FG injected near the motor cortex is detected in the VPL. Fi: Fimbria. IC: Internal capsule. RT: Thalamic reticular nucleus. VPL: Thalamic ventral posterolateral nucleus. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52932/52932fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions at JoVE.com

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

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Research

JoVE Journal

Neuroscience

14.2K Views.  University of Duisburg-Essen. In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Video: Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

Biology

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Medicine

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and autism. As evidence suggests that remyelination can effectively ameliorate the disease symptoms, there is an increasing focus on drug development to promote the myelin regeneration process. Thus, a region-selectable and result-reliable drug delivery technique is required to test the efficiency and specificity of these drugs in vivo. This protocol introduces the osmotic pump implant as a new drug delivery approach in the lysolecithin-induced demyelination mouse model. The osmotic pump is a small implantable device that can bypass the blood-brain barrier (BBB) and deliver drugs steadily and directly to specific areas of the mouse brain. It can also effectively improve the bioavailability of drugs such as peptides and proteins with a short half-life. Therefore, this method is of great value to the field of central nervous system myelin regeneration research.

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and ...

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits is causal remains elusive. Moreover, whether these deficits are mediated by an increase in glucose levels in cerebrospinal fluid (CSF) and/or blood is also unclear. There are very few studies investigating the direct effects of high CSF glucose levels on central nervous system (CNS) function, especially on learning and memory, since current diabetes models are not sufficiently developed to address such research questions. This article describes a method to chronically increase CSF glucose levels for 4 weeks by continuously infusing glucose into the lateral ventricle using osmotic minipumps in mice. The protocol was validated by measuring glucose levels in CSF. This protocol increased CSF glucose levels to ~328 mg/dL after infusion of a 50% glucose solution at a 0.25 &#181;L/h flow rate, compared to a CSF glucose concentration of ~56 mg/dL in mice that received artificial cerebrospinal fluid (aCSF). Furthermore, this protocol did not affect blood glucose levels. Therefore, this method can be used to determine the direct effects of high CSF glucose on brain function or a specific neural pathway independently of changes in blood glucose levels. Overall, the approach described here will facilitate the development of animal models for testing the role of high CSF glucose in mediating features of Alzheimer&#39;s disease and/or other neurodegenerative disorders associated with diabetes.

This article describes a detailed protocol to increase glucose concentration in the cerebrospinal fluid (CSF) of mice. This approach can be useful for studying the effects of high CSF glucose on neurodegeneration, cognition, and peripheral glucose metabolism in mice.

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits ...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Source: Ajoy, R., Chou, S. Y. <a href="https://www.jove.com/v/56410">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the assembly of a micro-osmotic pump brain infusion system followed by its surgical implantation in the mouse skull. This system enables continuous delivery of potential drugs and facilitates the study of their effects on the brain and brain tissue.

Source: Ajoy, R., Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

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