Name: osmotic minipump implantation for increasing glucose concentration in mouse cerebrospinal fluid
Uploaded: 2023-04-07T14:20:00
Description: Watch this Scientific Journal Video about Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid at JoVE.com

National Institutes of Health grant DK124619 to KHC.
Start-up funds and pilot research award, Department of Medicine, University of Rochester, NY, to KHC.
The Del Monte Institute for Neuroscience Pilot Research Award, University of Rochester, to KHC.
University Research Award, Office of the Vice President for Research, University of Rochester, NY, to KHC.
MUR designed and performed the method, analyzed results, prepared graphs and figures, and wrote and edited the manuscript. KHC conceived and supervised the study, analyzed results, and wrote and edited the manuscript. KHC is the guarantor of this work. All authors approved the final version of the manuscript.

All mouse procedures were approved by the Institutional Animal Care and Use Committee at the University of Rochester and were performed according to the US Public Health Service guidelines for the humane care and use of experimental animals. Six weeks old C57BL/6J male mice used for this study were commercially obtained. All the animals were group housed (5 mice per cage) in a room with a 12 h day/night cycle and were given access to food and water ad libitum. After the mice were implanted with a cannula for inf...

<ol>
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Molecular Basis of Disease. 1863 (1), 266-273 (2017).</li><li>Ernst, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+db/db+mice+exhibit+central+nervous+system+and+peripheral+molecular+alterations+as+seen+in+neurological+disorders.">Diabetic db/db mice exhibit central nervous system and peripheral molecular alterations as seen in neurological disorders.</a> Translational Psychiatry. 3 (5), 263 (2013).</li><li>Wang, Y., Yang, Y., Liu, Y., Guo, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+impairments+in+type+1+diabetes+mellitus+model+mice+are+associated+with+synaptic+protein+disorders.">Cognitive impairments in type 1 diabetes mellitus model mice are associated with synaptic protein disorders.</a> Neuroscience Letters. 777, 136587 (2022).</li><li>Jolivalt, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes+exaggerates+features+of+Alzheimer's+disease+in+APP+transgenic+mice.">Type 1 diabetes exaggerates features of Alzheimer's disease in APP transgenic mice.</a> Experimental Neurology. 223 (2), 422-431 (2010).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Paxinos and Franklin's The mouse brain in stereotaxic coordinates. , (2019).</li><li>Vinik, A. I., Maser, R. E., Mitchell, B. D., Freeman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+autonomic+neuropathy.">Diabetic autonomic neuropathy.</a> Diabetes Care. 26 (5), 1553-1579 (2003).</li><li>Furman, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptozotocin-induced+diabetic+models+in+mice+and+rats.">Streptozotocin-induced diabetic models in mice and rats.</a> Current Protocols. 1 (4), 78 (2021).</li><li>Grieb, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+streptozotocin+injections+as+a+model+of+Alzheimer's+disease:+in+search+of+a+relevant+mechanism.">Intracerebroventricular streptozotocin injections as a model of Alzheimer's disease: in search of a relevant mechanism.</a> Molecular Neurobiology. 53 (3), 1741-1752 (2016).</li><li>Kealy, 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=Acute+inflammation+alters+brain+energy+metabolism+in+mice+and+humans:+role+in+suppressed+spontaneous+activity,+impaired+cognition,+and+delirium.">Acute inflammation alters brain energy metabolism in mice and humans: role in suppressed spontaneous activity, impaired cognition, and delirium.</a> The Journal of Neuroscience. 40 (29), 5681-5696 (2020).</li><li>Dougherty, J. 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This article reports a detailed protocol to increase CSF glucose in mice by using osmotic minipumps connected to a cannula implanted in the lateral ventricle. The chronic infusion of glucose in the mouse brain through this procedure&#160;will be useful in delineating the effects of long-term hyperglycorrhachia on cognition, systemic glucose metabolism, and energy balance&#160;and for better understanding the pathogenesis of diabetes complications.
Chronic diabetes causes brain damage that inte...

Both type 1 and type 2 diabetes impair brain function1,2,3. For example, diabetes increases the risk of cognitive decline and neurodegenerative disorders, including Alzheimer&#39;s disease3,4. Moreover, people with diabetes have defective glucose sensing in the brain5,6. This defect contributes to the pathogenesis of hypoglycemia associated unawareness and an insufficient counter-regulatory response to hypoglycemia7,8, which can be fatal if not treated immediately.
Considering that diabetes increases glucose levels in the blood as well as in cerebrospinal fluid (CSF)9, it is important to determine whether one or both of these factors contribute to impaired brain function. Whether diabetes causes brain damage by high CSF glucose alone or in combination with other factors like insulin deficiency or insulin resistance is also an open question. Animal models of type 1 and type 2 diabetes show cognitive decline and neurodegeneration in addition to an affected energy balance and peripheral glucose metabolism10,11,12,13. However, from these models, it is not feasible to uncouple the selective effects of high CSF glucose versus blood glucose levels in mediating the complications of diabetes on brain function.
This protocol describes methods to develop a mouse model of hyperglycorrhachia to test the effects of chronically high CSF glucose levels on brain function, energy balance, and glucose homeostasis. The mouse model developed through this technique presents a tool for studies investigating the etiological role of dysregulated glucose homeostasis on neural and behavioral function.
Therefore, the proposed approach will be useful in understanding the direct effects of elevated CSF glucose levels in various pathophysiological conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m syringe filter</td>
			<td>Membrane solutions</td>
			<td>SFPES030022S</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL sterile Syringe (Luer-lok tip)</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL TB syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Glass beaker</td>
			<td>Fisher&#160;</td>
			<td>N/a</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol (Koptec)</td>
			<td>DLI</td>
			<td>UN170</td>
			<td>Use 70% dilution to clean the surgery area</td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Fisher&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Allignment indicator</td>
			<td>KOPF</td>
			<td>1905</td>
			<td></td>
		</tr>
		<tr>
			<td>Alzet brain infusion kit</td>
			<td>DURECT</td>
			<td>Kit # 3; 0008851</td>
			<td>Cut tubing in the kit to 1 inch length</td>
		</tr>
		<tr>
			<td>Alzet osmotic pump</td>
			<td>DURECT</td>
			<td>2004</td>
			<td>Flow rate 0.25 &#181;L/h</td>
		</tr>
		<tr>
			<td>Anesthesia system</td>
			<td>Kent Scientific</td>
			<td>SomnoSuite</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Avrio Health</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 . 2H2O</td>
			<td>Fisher&#160;</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula holder</td>
			<td>KOPF</td>
			<td>1966</td>
			<td></td>
		</tr>
		<tr>
			<td>Centering scope</td>
			<td>KOPF</td>
			<td>1915</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Cement Liquid</td>
			<td>Lang Dental</td>
			<td>REF1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement Powder</td>
			<td>Lang Dental</td>
			<td>REF1220-C</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose&#160;&#160;</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric drill</td>
			<td>KOPF</td>
			<td>1911</td>
			<td>While drilling a hole avoid rupturing dura mater</td>
		</tr>
		<tr>
			<td>Eye lubricant (Optixcare)</td>
			<td>CLC Medica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bead sterilizer (Germinator 500)</td>
			<td>VWR</td>
			<td>101326-488</td>
			<td>Place instruments in sterile water to let them cool before surgery</td>
		</tr>
		<tr>
			<td>Glucose Assay Kit</td>
			<td>Cayman chemical</td>
			<td>10009582</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>H1009-500ml</td>
			<td>Apply 3% H2O2 on skull surface to make the cranial sutures visible.</td>
		</tr>
		<tr>
			<td>Hair Clipper</td>
			<td>WAHL</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Heatpax</td>
			<td>19520483</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Fluriso)</td>
			<td>Zoetis</td>
			<td>NDC1385-046-60</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>0395-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>WPI</td>
			<td>M1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifying desk lamp</td>
			<td>Brightech</td>
			<td>LightView Pro Flex 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal Spatula</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 . 6H2O</td>
			<td>Fisher&#160;</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator (Right handed)</td>
			<td>WPI</td>
			<td>M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator with digital display</td>
			<td>KOPF</td>
			<td>1940</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 . 7H2O</td>
			<td>Fisher&#160;</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 . H2O</td>
			<td>Fisher&#160;</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin</td>
			<td>Johnson &#38; Johnson</td>
			<td>N/A</td>
			<td>Apply topical oinment to prevent infection</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>DM-999</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl (Carprofen) 50mg/ml</td>
			<td>Zoetis</td>
			<td>N/A</td>
			<td>5 mg/kg, subcutaneous, for analgesia</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic allignment system</td>
			<td>KOPF</td>
			<td>1900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 27 gauge needle</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton tip applicators (Solon)</td>
			<td>AMD Medicom</td>
			<td>56200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile nylon sutures (5.0)</td>
			<td>Oasis</td>
			<td>MV-661</td>
			<td>Use non-absorable suture for closing the wound</td>
		</tr>
		<tr>
			<td>Sterile sharp scissors&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves (Nitrile)</td>
			<td>Ammex</td>
			<td>N/A</td>
			<td>Change gloves if there is suspision of contamination</td>
		</tr>
		<tr>
			<td>Tray</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Male mice were implanted with a cannula assembled to an osmotic minipump (Figure&#160;1) to chronically infuse aCSF or a 50% glucose solution into their lateral ventricles (Figure 2). CSF was collected 10 days after the surgery (Figure 3) to validate the efficacy of this procedure. The results showed an increase in the CSF glucose levels (mean: 327.7 mg/dL) in mice infused with 50% glucose compared to that (mean: 56.5 mg/dL) in mice...

Watch this Scientific Journal Video about Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid at JoVE.com

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

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 ...

This technique increases the Cerebrospinal Fluid or CSF glucose levels in mice without affecting their blood glucose concentration. This helps to investigate the direct effects of high CSF glucose levels on the animal's brain function. This technique can establish animal models to explore the theological role of higher CSF glucose on cognitive dysfunction.Leading to the discovery of new molecular targets to improve cognitive function. The method will also help determine whether high CSF glucose levels are sufficient to mediate phenotypes of neurodegenerative disorders in mice. Attention must be paid to the proper assembly and priming of the cannula setup and insertion of the cannula in the brain at the correct coordinates.To begin the surgery for Osmotic Minipump Implantation place the anesthetized mouse on the stereotaxic frame. Fit the animal's head on the incisor bar. Cover the nose with the nose cone to maintain the anesthesia and connect isofluorane flow to the nose cone at a rate of 1.5 to 2%Place a heating pad under the mouse to maintain the body temperature.After disinfecting the surgical area and making an incision expose the skull surface with a spatula and wipe the blood, if any, with cotton tips. Use a cauterizer if bleeding profusely. Rub with a cotton tip dipped in 3%hydrogen peroxide to expose the cranial sutures.Working under a microscope, take note of bregma and lambda to navigate the skull coordinates. Set all the coordinates to zero at bregma. Using an electric drill carefully and without rupturing the dura mater make a hole at the coordinates 0.5 millimeters posterior to bregma and 1 millimeter lateral to midline toward the right.Insert a hemostat and gently open and close it to make a pocket for the mini pump. Holding the pump from the tip of the flow modulator push it through the incised skin into the created pocket. Apply some glue on the underside of the cannula, and fix it in the cannula holder.Ensure that the cannula is not clogged and the solution is flowing properly. Then lower the cannula slowly through the drilled hole, two millimeters ventral to the skull surface. Leave it there for at least five minutes to allow the glue to solidify.With the help of scissors, clip the top of the cannula ensuring it doesn't dislodge from the skull surface. Cover the cannula with a layer of dental cement for extra support. For Cerebrospinal Fluid or CSF collection, place the properly anesthetized mouse on the stereotaxic frame.After fixing the head on the frame as demonstrated previously rotate the knobs to tilt the head so that the nose faces downward. After disinfecting the surgical area and making an incision remove the support from the mouse body and let the mouse rest vertically so that the neck is fully extended dorsally. Using curved blunt forceps, gently bisect the posterior neck muscles from the midline to create a small window.Then use a wet cotton tip applicator to gently displace the neck muscles from the midline to the periphery. Observe whether the cisterna magna is exposed as a triangular window with a transparent dura membrane. Place the micro manipulator assembly on the stereotaxic frame next to the mouse while ensuring the capillary tip does not touch any surface.Gently break the capillary tip without disturbing the setup. Rotate the corresponding knobs on the micro manipulator to align slowly and move the capillary tip toward the cisterna magna. Some resistance is felt once the capillary tip touches the cisterna magna membrane.Very slowly push the tip against the membrane with the help of the knobs being careful not to damage any blood vessels in the membrane. As the membrane is pierced, CSF flows into the capillary at once due to negative capillary pressure. Leave the setup for a few minutes until approximately 10 microliters of CSF collects in the capillary.Carefully remove the capillary from the syringe and attach it to a microcap bulb dispenser. Press the bulb to transfer the CSF into a sterile micro centrifuge tube. Place the support under the mouse and rotate the stereotaxic knobs to level the head.Close the wound with nylon sutures. Inject 300 microliters of sterile saline subcutaneously before removing the mouse from the apparatus. Give postoperative care to the animals until it is time to remove the sutures, about 7 to 10 days after surgery.CSF collected 10 days after the surgery showed about a sixfold increase in glucose level in mice, infused with 50%glucose compared to that in mice infused with CSF. However, the blood glucose levels were not different between the groups. While lowering the cannula in the brain, retract it back and check if CSF comes out of the hole.It is also vital to correctly identify cisterna magna and prevent contamination of CSF with blood. This technique can also be used to deliver any drug that is unable to cross the blood-brain barrier to test its effect on brain function. There is a very limited number of animal models of diabetes, and they disturb glucose homeostasis in the peripheral environment.This model, however, helps investigate brain specific changes in glucose regulation on the neural function.

osmotic-minipump-implantation-for-increasing-glucose-concentration

Research

JoVE Journal

Biology

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...