We thank Caroline Bouchard from the animal facility for the rat work. A.S. holds a J.A. De S&#232;ve master fellowship, and B.P. a COPSE fellowship from the Universit&#233; de Montr&#233;al This work was funded by grants attributed to J.B. from FRQS-Pfizer and start-up funds from H&#244;pital du Sacr&#233;-Coeur de Montr&#233;al Research Center.

Ethics statement: The animal protocol for this project obtained the approval from the Animal Care Committee of the H&#244;pital du Sacr&#233;-Coeur de Montr&#233;al in compliance with the guidelines of the Canadian Council on Animal Care.
1. Catheter Preparation before Stereotaxic Surgery
<ol>
	<li>Cut PE50 catheters (6 cm in length) that will be used to connect cannula with osmotic pumps (see Figure 1A). Fill PE50 catheters with artificial cerebrospinal fluid (aCSF) and seal both ends of the catheters. Keep the catheter at 4 &#176;C until used.</li>
</ol>
2. Cannula Implantation by Stereotaxy
<ol>
	<li>Perform the surgery in sterile conditions. Sterilize all the surgical instruments and materials by autoclaving. Clean the stereotaxic apparatus and the working area thoroughly, and disinfected with a 70% ethanol solution. Wear a surgical mask, hair bonnet and sterile gloves.</li>
	<li>Anesthetize rats by injecting intraperitoneally (i.p.) a solution of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). Confirm anesthesia by checking movement after a gentle toe pinch.</li>
	<li>Inject a non-steroidal anti-inflammatory drug (Meloxicam; 1 mg/kg) subcutaneously (s.c.) to the anesthetized-animal at least 30 min prior to surgery.</li>
	<li>Shave the head of the animal using clippers and disinfect the skin with a solution of chlorhexidine gluconate 2% and isopropyl alcohol 2% three times. Apply veterinary ophthalmic ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Place the animal on a stereotaxic frame with the ear bars. Fix one cannula on the holder arm of the stereotaxic frame. Inject (s.c.) a local anesthetic agent (bupivacaine (1.5 mg/kg) and lidocaine (1.5 mg/kg) on the top of the head.&#160;Anesthesia is maintains during the whole procedure by placing a nose cone delivering 3% isoflurane. The entire surgical field should be draped off, but it was not done here to better demonstrate the technique.</li>
	<li>Make an incision of 3 cm on the top of the head with a scalpel. Install 4 clamps around the incision to leave the skull clear. Using a round-tip scissor, make a pocket (2 x 2 cm2) under the skin between shoulder blades of the animal.</li>
	<li>Scrape&#160;the periosteum of the skull with a blade. Apply a gauze pad on the skull if bleeding.</li>
	<li>Verify that the skull is flat and well aligned on the stereotaxic frame. To make sure that the skull is flat, check the height coordinates at the bregma and at the lambda. Take the coordinates of the bregma that will be used as the reference point.</li>
	<li>Starting from the bregma, calculate the coordinates of the two guide cannula that will be implanted bilaterally in the hippocampus (anteroposterior: - 0.42 cm; mediolateral: &#177; 0.30 cm; dorsoventral: - 0.28 cm according to the Paxinos and Watson rat brain atlas)29. Indicate the position of the cannula with a marker.</li>
	<li>Drill a hole (0.5 mm) in the skull at the implantation point of both cannula. Drill two other holes approximately 5 mm above and below those points to insert screws that will solidify cannula when applying dental cement.</li>
	<li>Cut one end of the catheter previously filled with aCSF with a scalpel, and insert it in the angle arm of the cannula. Fix the first cannula with dental cement. Avoid putting dental cement around the position on the second cannula.</li>
	<li>Let the dental cement dry for 2 - 3 min, then remove the holder arm from the first cannula, and fix the second cannula in it. Repeat steps 2.11 and 2.12. Put dummy cannula on both guide cannula. Ensure that the dummy and guide cannula are of the same length to prevent tissue infiltration and blocking the cannula.</li>
	<li>Insert the free end of both catheters in the pocket previously made between shoulder blades of the animal. Remove the clamps and stitch the skin with a suture thread 4-0. 
	NOTE: The top guide cannula needs to be accessible for upcoming A&#946;o infusions.</li>
	<li>Remove the animal from the stereotaxic frame and put it back in its cage. During post-surgical recovery, place the cage on a heating water blanket until the animal wakes up. The cage should be partially on the pad so the rat can move away from the heat if needed.&#160;&#160;Monitor the animal constantly until it regains sufficient consciousness to maintain sternal recumbency.</li>
	<li>Return rats to the animal facility to recover from the surgery for 10 days under close monitoring. 
	NOTE: Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Meloxicam (1 mg/kg) is given once per day during two days following surgery to treat pain.</li>
</ol>
3. Osmotic Pump Installation
<ol>
	<li>One day before installation, fill osmotic pumps with 6E10 antibody (1 mg/ml; 100 &#181;l) and control IgG1 antibody (1 mg/ml; 100 &#181;l) according to the manufacturer&#39;s instructions under sterile condition. Keep the pumps in sterile distilled water at 37 &#176;C overnight to activate pumps.</li>
	<li>Anesthetize rats with isoflurane 3% to install osmotic pumps (0.5 &#181;l/hr for 7 days). Shave between shoulder blades and disinfect the skin with a solution of chlorhexidine gluconate 2% and isopropyl alcohol 2%. Make an incision of 2 cm with a scalpel between shoulder blades to locate the PE50 catheters connected to the guide cannula.</li>
	<li>Cut the end of the PE50 catheters containing dental cement with a scalpel. Connect the osmotic pumps to the PE50 catheters, and add some dental cement at the pump and catheter junction to secure the connection.</li>
	<li>Stitch the skin tightly with a suture thread 4-0. Put the animal back in its cage on a heating water&#160;pad. Observe the animal wake up rapidly from an isoflurane anesthesia (about 5 min). 
	NOTE: The surgery takes approximately 5 to 10 min.</li>
</ol>
4. A&#946;o Infusions in Awake and Freely Moving Rats
<ol>
	<li>Prepare the A&#946;o solution (0.2 &#181;g/&#181;l) as previously reported.28,30 Allow A&#946;o to aggregate dynamically and spontaneously for 1 hr at room temperature before infusion.</li>
	<li>Install two 10 &#181;l syringes on an infusion pump. Fill the syringes with 5 &#181;l of sterile distilled water. Cut two PE50 catheters to about 60 cm in length with a scalpel. Fill both catheters with sterile distilled water using 1 ml syringes and 21G needles.</li>
	<li>Keep the 1 ml syringes at one end of the catheter and connect the other end to the Hamilton syringes. Remove the 1 ml syringes and check that there is no air bubbles within the catheters.</li>
	<li>Insert internal cannula at the end of PE50 catheters. Using sterile distilled water, fill the internal cannula connected to PE50 catheters up to1 &#181;l on Hamilton syringes. Make an air bubble by pulling back the pistons up to 2 &#181;l.</li>
	<li>Mix the A&#946;o solution by pipetting up and down using a tip of minimum of adherence. Avoid forming bubbles during mixing. Fill both internal cannula with 1.5 &#181;l of A&#946;o solution by pulling back the pistons up to 3.5 &#181;l.</li>
	<li>Make lines with a marker before and after the air bubble done in both catheters. This serves as a check point of ongoing infusion. For infusion, bring the cage near the infusion pump and remove its cover.</li>
	<li>Place the rat in a snuggle and tightly scrape&#160;the arm of the snuggle around its neck to immobilize the head of the rat. Remove dummy cannula from the two guide cannula. Insert the internal cannula previously prepared in the guide cannula. Verify that they are fully inserted and well-fixed to the base of the guide cannula.</li>
	<li>Release rat from the snuggle and put it back in its cage to limit contention stress. Monitor closely to ensure that the catheters do not twist together and the infusion is done properly.</li>
	<li>Turn on the infusion pump. Inject 1 &#181;l of A&#946;o solution at a rate of 0.1 &#181;l/min (10 min). During infusion, check that the syringe piston moves from 3.5 &#181;l to 2.5 &#181;l, and that the air bubble in both PE50 catheters move continuously.</li>
	<li>Leave the internal cannula in place for another 5 min after infusion to allow efficient diffusion of A&#946;o solution. Bring the rat back in the snuggle to remove internal cannula and capped guide cannula to prevent reflux of the injected solution. Use dummy cannula that stop just before the angle arm of the cannula. Return the animal in its cage.</li>
	<li>Repeat A&#946;o infusion once per day over 6 consecutive days. Euthanize the animal by decapitation.</li>
</ol>

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Publication of this video-article is sponsored by ALZET Osmotic Pumps.

There are critical steps within this protocol that required special attention. When implanting the cannula, avoid putting dental cement when it is too liquid to prevent blocking the hole of the second cannula. It is important to place dental cement at the free end of the P50 catheter attached to the pump to prevent irritation and a possible inflammatory response. The day of the stereotaxic surgery, use dummy cannula that are the same length as guide cannula to avoid blocking cannula. However, after installing pumps use shorter dummy cannula that stop before the angle arm of the cannula to allow proper infusion of the solution from the pump to the hippocampus. Monitor closely A&#946;o infusions and verify that the air bubble done in catheters is moving continuously during infusions. Always make sure that the injecting cannula is completely inserted into the guide cannula during infusions.
If problem is encounter during A&#946; infusion, verify that the internal cannula is not blocked. If it is the case, flush sterile distilled water through the internal cannula. If the guide cannula is obstructed, turn the internal cannula into the guide cannula. Otherwise move the internal cannula up and down. Contention in the snuggle can be stressful for rats, especially on the first day. To decrease the stress of the animal, we recommend to manipulate and habituate rats to the snuggle before the stereotaxic surgery.
Many advantages can be attributed to this novel and flexible in vivo approach. Indeed, the nature of A&#946;o injected can be accurately control before infusion, and different type of A&#946; preparations (for example synthetic vs brain-derived A&#946; solutions) can be injected to evaluate their neurotoxicity in vivo. This model can also be used to investigate mechanisms by which various A&#946; species (e.g., monomers, low- and high-molecular-weight oligomers, protofibrils) can induce neurotoxic effects in vivo, and how treatments like immunotherapy might counteract their deleterious impact in the brain. Since the infusions are perform in awake, freely-moving animals, there are no confounding effects between anesthetic agents and the A&#946;o solution on signaling pathways, as shown in previous studies.32,33 Infusions in freely moving animal are also compatible with behavioral testing any time before and after the infusions.
Infusions of A&#946;o and pump installation can be done in animal of different ages to determine the effects of A&#946;o and treatments during aging. Since neurodegeneration occurs in vicinity of the infusion site, synapse and neuronal loss can be induced in different and localized brain regions. The collateral infusion of A&#946;o and control (vehicle or scramble A&#946;) allows controlling for any change within the same animal. Conversely, A&#946;o or control solutions can be injected bilaterally in the right and left hippocampus, for example when testing animals in behavioral tasks. Infusion of A&#946;o and treatment can be done simultaneously or alternatively pumps can be installed after A&#946;o infusion to evaluate if the treatment is effective after A&#946; deposition. The same protocol described here can also be used when doing intracerebroventricular infusions of A&#946;o. The effect of A&#946;o on intracellular signaling pathways can be evaluated before and after neuronal loss within a reasonably short time frame. The dose and number of A&#946; infusions can also be adjusted to obtain a more or less severe A&#946; pathogenicity.
Although very versatile, this technique has some limitations. Cannula implantation produces a mechanical disruption of the tissue and neuroinflammation in the first few days following surgery. Thus, it is essential to wait at least one week after surgery before starting A&#946;o infusion, and to add proper controls (injection of vehicle or inactive scramble A&#946;) to take into account these events. Also, only a small volume of A&#946;o can be infused to limit diffusion of the solution.
The osmotic pumps represent a convenient and unique delivery method for preclinical validation of agents designed to prevent A&#946;-induced neurodegeneration. Since immunotherapy with the 6E10 antibody has been shown previously to decrease A&#946; accumulation in the brain,31 we used the 6E10 antibody as a proof-of-concept to validate our new in vivo approach. The used of osmotic pumps in this model might now be used to develop novel disease modifying therapies that could prevent the deposition and neurotoxicity of A&#946;o in pre-clinical AD patients.

It was initially proposed that accumulation of insoluble A&#946; species in the brain was central to AD pathogenesis.1,2 However, amyloid plaques are also detected in some cognitively normal elderly.3-7 To overcome the poor correlation existing between plaque depositions and cognitive deficits in AD, latest reports have shown the presence of toxic soluble A&#946;o at the onset of the disease, which correlate much better with the clinical state of the patients.8-14 Since the process of A&#946; oligomerization is very dynamic, it was suggested that neurotoxicity is induced by various A&#946;o instead of only one specific type of oligomer.14-16 Since many studies have shown that A&#946;o can initiate synapse dysfunctions prior to synapse and neuronal loss,17-24 current theories indicate that A&#946;-related treatment might be effective in early AD rather than at later stages as tested so far in clinical trials.
One hallmark feature of AD pathogenesis is the massive and widespread cell death observed in the late stages of the disease, and the significant synapse and neuronal loss observed in localized brain regions when memory deficits become detectable at the clinical level. The perforant pathway that projects from the entorhinal cortex (EC) to the dentate gyrus (DG) is perturbed markedly at the early onset of AD.25,26 During the prodromal state of AD when mild cognitive impairment (MCI) becomes apparent significant cell death is detected in the EC as well as synaptic loss in the DG.25,26
Although a large body of evidence has pinpointed the toxic action of soluble A&#946;o in early AD,8-14 A&#946;o-induced neurodegeneration observed in neuronal culture or organotypic brain slice culture has been more challenging to reproduce in animal models.27 Most of the transgenic AD models overexpressing A&#946; have amyloid plaques, tau hyperphosphorylation, synaptic deficiency and memory deficits.27 However, these models have been much less successful in modelling cell death observed in the hippocampus of AD patients. To overcome these technical issues we developed a model based on intracerebral infusions of soluble A&#946;o. We reported earlier that repeated hippocampal infusions of soluble A&#946;o induce gradual neuronal loss and tau hyperphosphorylation, two pathological hallmarks associated with memory decline in AD.28 Here, we are showing a novel method to test AD therapies using cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) with osmotic pumps.
Osmotic pumps provide a unique way to test in vivo the efficiency of any antibody (or other compounds) against A&#946;o-induced neurodegeneration directly at the infusion site of A&#946;o. Thus, these pumps represent a convenient tool to establish a solid proof-of-concept regarding the mechanisms of action of potential therapeutic agents in AD. Since recent reports point out the critical impact of soluble A&#946;o in the early stages of AD, many treatments directed toward A&#946;o are actually being tested by academic and pharmaceutical laboratories. This novel animal model allows mimicking the synaptic and neuronal loss observed in early AD, and osmotic pumps are used to infuse continuously treatment agents specifically at the A&#946;o infusion site. The repetitive failures of AD therapies tested over the last few years in mild to moderate patients as prompted researchers to initiate trials in pre-dementia patients before A&#946;o begin to accumulate abundantly and generate irreversible brain damage. In this context, testing new compounds that prevent the deposition and consequently the neurotoxicity of A&#946;o might be of interest in pre-clinical patients.

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			<td height="21" style="height:21px;width:279px;">Artificial Cerebrospinal Fluid (aCSF)</td>
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			<td height="21" style="height:21px;">Guide Cannula</td>
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			<td height="21" style="height:21px;width:279px;">Alzet osmotic pumps model 1007D</td>
			<td style="width:167px;">Durect corporation</td>
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			<td style="width:147px;">A-1163-1</td>
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			<td height="21" style="height:21px;width:279px;">Hamilton syringe (10 &#181;L)</td>
			<td style="width:167px;">Fisher Scientique</td>
			<td style="width:147px;">14815279</td>
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			<td style="width:167px;">Harvard Apparatus</td>
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			<td height="21" style="height:21px;width:279px;">Syringe 1 mL</td>
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neurodegeneration in an animal model of chronic amyloid-beta oligomer infusion is counteracted by antibody treatment infused with osmotic pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

The neurotoxic effect of A&#946;o was investigated in Long-Evans rats by implanting cannula in the DG of the hippocampus. Soluble A&#946;o were injected every day over six consecutive days. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of 6E10 or control IgG1 antibody in the hippocampus using osmotic pumps (Figure 1A). For immunohistochemistry rats were anesthetized, perfused transcardially with 0.9% NaCl, and brains immersed in 4% paraformaldehyde solution for 48 hr and 15% sucrose solution for 24 hr. Free-floating coronal sections (40 &#181;m) were treated with 0.3% H2O2 for 30 min, blocked in 1% goat serum for 1 hr, and incubated overnight at 4 &#176;C with an anti-pan-A&#946; antibody. Sections were treated with 70% formic acid for 3 min before applying the antibody. Detection was performed using the ABC complex and a 0.05% 3,3-diaminobenzidine solution. Sections were mounted on gelatin-coated glass slides, air-dried 2 hr, dehydrated (30, 70, 95, and 100% alcohol), incubated in toluene 5 min, and coverslipped. For cresyl violet staining, sections were incubated 10 min in a 0.5% cresyl violet solution, incubated 1 min in a 0.5% acetic acid solution, decolored (70, 95, and 100% alcohol), immersed in toluene 5 min, and covered with glass coverslip.
The results presented here show the deposition of A&#946;o in the DG in vicinity of the infusion site, and cell death associated with this accumulation. We found that the level A&#946;o was substantially decreased by 6E10 antibody treatment (Figure 1B). Marked neurodegeneration was observed near the injecting site of A&#946;o, and was attenuated by 6E10 antibody treatment (Figure 1B). These results are consistent with A&#946;o accumulation that we observed in the DG following repeated A&#946;o infusions in mice.28 Clearance of amyloid deposition by immunotherapy with 6E10 antibody shown here is in line with another report done in a transgenic AD mouse model.31
<img alt="Figure 1" src="/files/ftp_upload/54215/54215fig1.jpg" /> 
Figure 1. Photo of the Animal with Bilateral Cannula During A&#946;o Infusion. A solution of A&#946;o (0.2 &#181;g/&#181;l; 1 &#181;l) was injected in awake and freely moving rats (once a day for 6 days) using PE50 catheters connected to internal cannula inserted into guide cannula. Treatment with control (Ctl) IgG1 or 6E10 antibody was infused directly at the site of A&#946;o infusion using osmotic pumps located subcutaneously between the shoulder blades of the animal. <a href="https://www.jove.com/files/ftp_upload/54215/54215fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54215/54215fig2.jpg" /> 
Figure 2. Neuronal Loss Induced by A&#946;o Deposition is Attenuated by 6E10 Antibody Treatment. A, A&#946;o (0.2 &#181;g/&#181;l; 1 &#181;l) was injected once a day during 6 consecutive days, and treated with Ctl (IgG1) or 6E10 antibody at the site of infusion using osmotic pumps. B, Representative accumulation of A&#946;o in the DG on a section immediately next to cannula insertion, and representative staining with cresyl violet showing cell loss. Scale bars: 50 &#181;m (n = 4) <a href="https://www.jove.com/files/ftp_upload/54215/54215fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps at JoVE.com

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

neurodegeneration-an-animal-model-chronic-amyloid-beta-oligomer

Research

JoVE Journal

Medicine

9.2K Views.  Université de Montréal. To study the effects of Aβo in vivo, we developed a model based on repeated hippocampal infusions of soluble Aβo coupled with continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of Aβo.

Video: Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

The Miniature Pig: A Large Animal Model for Cochlear Implant Research

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, no standard model exists for investigating the electrophysiology and histopathology in patients or animal models with a CI or for evaluating new models of electrode arrays. A large animal model with cochlea characteristics similar to those of humans may provide a research and evaluation platform for advanced and modified arrays before their use in humans. 
To this end, we established standard CI methods with Bama mini-pigs, whose inner ear anatomy is highly similar to that of humans. Arrays designed for human use were implanted into the mini pig cochlea through a round window membrane, and a surgical approach followed that was similar to that used for human CI recipients. Array insertion was followed by evoked compound action potential (ECAP) measurements to evaluate the function of the auditory nerve. This study describes the preparation of the animal, surgical steps, array insertion, and intraoperative electrophysiological measurements. 
The results indicated that the same CI used for humans could be easily implanted in mini-pigs via a standardized surgical approach and yielded similar electrophysiological outcomes as measured in human CI recipients. Mini-pigs could be a valuable animal model to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches before applying them to human beings.

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, ...

Mongolian Gerbils as an Animal Model of Wound Healing

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving patients' eye health. Historically, corneal healing has been studied in rodents such as mice and rats, but these models might not completely mimic human disorders. However, information on other rodents such as Mongolian gerbils (Meriones unguiculatus) is scant in corneal research.
Here, we describe a technique to develop a novel animal model for studying corneal healing after photorefractive keratectomy. Due to the limited literature available on the cornea of M. unguiculatus, we also describe a histological analysis of the normal cornea. These research techniques can also be employed in the study of eye diseases because of the similarity between the corneas of Mongolian gerbils and humans in terms of genetics, anatomy, and physiology.

This article describes a new animal model developed to study the anatomy and histology of the cornea and its healing processes. This new animal model uses the Mongolian gerbil, which has a cornea with many similarities to the human cornea.

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving ...

3D Reconstruction for the Diagnosis and Treatment of Pulmonary Nodules

A 3D Digital Model for the Diagnosis and Treatment of Pulmonary Nodules

The three-dimensional (3D) reconstruction of pulmonary nodules using medical images has introduced new technical approaches for diagnosing and treating pulmonary nodules, and these approaches are progressively being acknowledged and adopted by physicians and patients. Nonetheless, constructing a relatively universal 3D digital model of pulmonary nodules for diagnosis and treatment is challenging due to device differences, shooting times, and nodule types. The objective of this study is to propose a new 3D digital model of pulmonary nodules that serves as a bridge between physicians and patients and is also a cutting-edge tool for pre-diagnosis and prognostic evaluation. Many AI-driven pulmonary nodule detection and recognition methods employ deep learning techniques to capture the radiological features of pulmonary nodules, and these methods can achieve a good area under-the-curve (AUC) performance. However, false positives and false negatives remain a challenge for radiologists and clinicians. The interpretation and expression of features from the perspective of pulmonary nodule classification and examination are still unsatisfactory. In this study, a method of continuous 3D reconstruction of the whole lung in horizontal and coronal positions is proposed by combining existing medical image processing technologies. Compared with other applicable methods, this method allows users to rapidly locate pulmonary nodules and identify their fundamental properties while also observing pulmonary nodules from multiple perspectives, thereby providing a more effective clinical tool for diagnosing and treating pulmonary nodules.

Author Spotlight: A 3D Digital Model for the Diagnosis and Treatment of Pulmonary Nodules  

The objective of this study is to develop a novel 3D digital model of pulmonary nodules that serves as a communication bridge between physicians and patients and is also a cutting-edge tool for pre-diagnosis and prognostic evaluation.

The three-dimensional (3D) reconstruction of pulmonary nodules using medical images has introduced new technical approaches for diagnosing and treating ...