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 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 µL/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's disease and/or other neurodegenerative disorders associated with diabetes.
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'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.
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 infusing glucose into the lateral ventricle, they were single housed to prevent any damage to the implants from other mice.
1. Assembly of osmotic minipumps
2. Surgery to implant osmotic pumps
3. Replacement of the minipumps
NOTE: Since, the minipumps used in this study last only for 4 weeks, the replacement of minipumps was also tested to extend the duration of glucose infusion, as it may be required in the case of long-term studies. This involved the following steps.
4. CSF collection procedure
5. Glucose assay
6. Blood glucose assay
Male mice were implanted with a cannula assembled to an osmotic minipump (Figure 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 infused with aCSF. This is about a six fold increase in the CSF glucose levels in the experimental mice compared to their control littermates (Figure 4A). The blood glucose levels were not different between the groups (Figure 4B).
Figure 1: Assembly of osmotic minipumps. (A) Infusion assembly with a cannula connected to a minipump through tubing. These pumps require at least 48 h to prime. (B) The presence of air bubbles outside the minipumps confirms the priming. Please click here to view a larger version of this figure.
Figure 2: Stereotaxic apparatus and accessories. (A,B) Stereotaxic equipment with an attached micromanipulator and other accessories. (C) Burr hole coordinates to insert the cannula. (D) Osmotic mini pump implantation, (E,F) Insertion of the cannula in the drilled hole. Maintain aseptic conditions throughout the surgery. Please click here to view a larger version of this figure.
Figure 3: Cerebrospinal fluid (CSF) collection procedure. (A) The dorsal neck muscles were gently displaced with blunt forceps to expose the cisterna magna. A 1 mm capillary with a 0.5 mm diameter tip was used to (B) rupture and (C,D) collect CSF from the cisterna magna. Please click here to view a larger version of this figure.
Figure 4: Measurement of glucose. (A) Increased CSF glucose (B) without affecting non-fasting blood glucose levels in mice infused with 50% glucose solution in the lateral ventricle. The efficacy of this protocol was validated by measuring CSF and blood glucose concentration 10 days after initiating the glucose infusion. Mice infused with 50% glucose solution had CSF glucose levels of 327.7 ± 30.1 mg/dL (mean ± standard error of mean) compared to mice that received artificial CSF infusion that had glucose levels of 56.5 ± 2.6 mg/dL. ****p < 0.0001, unpaired t-test. Error bars represent standard error of mean (n = 5). Please click here to view a larger version of this figure.
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 will be useful in delineating the effects of long-term hyperglycorrhachia on cognition, systemic glucose metabolism, and energy balance and for better understanding the pathogenesis of diabetes complications.
Chronic diabetes causes brain damage that interrupts the communication between the brain and the peripheral organs15. Diabetes also increases the risk of neurodegenerative diseases, including Alzheimer's disease3,4. Streptozotocin (STZ)-induced type 1 diabetes has been the standard rodent model in diabetes research16; STZ damages β-cells in the pancreas, leading to type 1 diabetes-like pathology. Further, in a modified version, the use of STZ accompanied with nicotinamides can induce type 2 diabetes. Another way of developing type 2 diabetes-like phenotypes in animals is through feeding them a high fat diet16. However, in the context of studying the effect of hyperglycemia on brain function, these techniques are limited in controlling for a large number of factors (e.g., peripheral insulin/glucagon levels, and metabolic function in general). Thus, any effect of STZ-induced diabetes on brain function can only be interpreted as an associated complication, instead of pinpointing a single etiological factor. Acute injection or chronic infusion of substances in the cerebroventricular space is a technique often used to test their direct effects on brain function. Intracerebroventricular (ICV) injection of STZ has been used to develop a rodent model of Alzheimer's disease, however, it remains uncertain whether STZ-associated neural damage is due to dysregulation in glucose sensing/homeostasis or other independent mechanisms, like STZ-induced oxidative stress and DNA damage17.
The procedures described in the current protocol will be useful in developing rodent models that can answer research questions, like whether an increase in CSF glucose concentration can cause cognitive impairment. The protocol described here could be used in determining the direct effects of high CSF glucose levels on the hypothalamus and hippocampus, among other brain regions involved in nutrient sensing, metabolism, and/or cognition. This method would also clarify whether an increase in CSF glucose levels affects insulin sensitivity, insulin secretion, food intake, and/or energy balance at baseline and in response to metabolic insults. Moreover, the protocol reported here would be applicable in testing hypotheses that require longitudinal studies. For example, data could be collected before, during, and at the end of the glucose infusions to compare findings from the same animals at different times. Such a strategy would address whether complications arising from high CSF glucose level are reversible after the normal CSF glucose level is restored. In contrast, the method could also be used for hypothesis-generating studies. For example, CSF could be collected from the same animals at different times and subjected to metabolomics or proteomics analysis to identify biomarkers or any metabolic insults produced by high level of CSF glucose. Similarly, different regions of the brain could be analyzed by spatial transcriptomics to yield cell-specific information that may have been altered by high CSF glucose.
The rationale for infusing glucose-free aCSF to a sham group was to keep the CSF glucose concentration at the baseline level, so that any change in CSF glucose level induced by cannula implantation can be naturally controlled. The results in this study showed that the sham group had a CSF glucose concentration of ~60 mg/dL (~3 mM), which is in the normal CSF glucose range in mice18. The CSF glucose levels observed in individuals with type 2 diabetes are ~110 mg/dL or ~6 mM9. In the current study, ICV infusion of 50% glucose at a rate of 125µg/h elevated CSF glucose levels to ~300 mg/dL (16 mM), which is supraphysiological19. Although this supraphysiological level of CSF glucose may not be clinically relevant to the levels observed in individuals with type 2 diabetes, the results presented in this study show that the infusion of glucose in CSF can induce a chronic elevation of CSF glucose concentration in mice.
The method presented here has some limitations. It involves sophisticated mouse brain surgery that requires relevant training, skills, and experience in performing such advanced procedures. Because the catheter and minipumps are implanted for the long term, meticulous care of the mice throughout the study is necessary to monitor for health concerns or damage to the catheter assembly. A 50% glucose concentration was selected because the viscosity of a solution beyond this concentration might have affected the infusion of glucose into the ventricles. The minipumps used in this protocol had a flow rate of 0.25 µL/h, so the group of mice with 50% glucose infusion received glucose at a rate of 125 µg/h, or 3 mg of glucose per day. This dose of glucose per unit time was therefore limited by the flow rate of the minipumps.
In summary, this article reports a validated method for the chronic increase of CSF glucose in mice. The information obtained from this model will be useful in determining whether or how an increase in CSF glucose levels is involved in mediating diabetes-associated complications, such as neurodegenerative disorders, or causing peripheral metabolic insults in diabetes and obesity.
Troubleshooting
If the tubing comes off of the cannula in the mice, a small amount of glue on the cannula-tubing connection can be applied while assembling the minipump. If stitches come off and the cannula becomes visible, the incision area can be completely closed by using sutures or staples. Nails from the hind paws of the mouse should be trimmed, so that there is a lower possibility of scratching the surgery area by the mouse. Moreover, be careful not to tie off the sutures so tight that the skin will rip, as mice have delicate skin.
For fast recovery after CSF collection, the injection of 300 µL of sterile saline subcutaneously after surgery is recommended. Furthermore, keeping the maximum volume of CSF collection to 10 µL is also important.
The authors have nothing to disclose.
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.
0.22 µm syringe filter | Membrane solutions | SFPES030022S | |
1 mL sterile Syringe (Luer-lok tip) | BD | 309628 | |
1 mL TB syringe | BD | 309659 | |
100 mL Glass beaker | Fisher | N/a | |
100% Ethanol (Koptec) | DLI | UN170 | Use 70% dilution to clean the surgery area |
50 mL conical tube | Fisher | N/A | |
Allignment indicator | KOPF | 1905 | |
Alzet brain infusion kit | DURECT | Kit # 3; 0008851 | Cut tubing in the kit to 1 inch length |
Alzet osmotic pump | DURECT | 2004 | Flow rate 0.25 µL/h |
Anesthesia system | Kent Scientific | SomnoSuite | |
Betadine solution | Avrio Health | N/A | |
CaCl2 . 2H2O | Fisher | C79-500 | |
Cannula holder | KOPF | 1966 | |
Centering scope | KOPF | 1915 | |
Dental Cement Liquid | Lang Dental | REF1404 | |
Dental cement Powder | Lang Dental | REF1220-C | |
D-glucose | Sigma | G8270 | |
Electric drill | KOPF | 1911 | While drilling a hole avoid rupturing dura mater |
Eye lubricant (Optixcare) | CLC Medica | N/A | |
Glass Bead sterilizer (Germinator 500) | VWR | 101326-488 | Place instruments in sterile water to let them cool before surgery |
Glucose Assay Kit | Cayman chemical | 10009582 | |
H2O2 | Sigma | H1009-500ml | Apply 3% H2O2 on skull surface to make the cranial sutures visible. |
Hair Clipper | WAHL | N/A | |
heating pad | Heatpax | 19520483 | |
Hemostat | N/A | N/A | |
Isoflurane (Fluriso) | Zoetis | NDC1385-046-60 | |
KCl | VWR | 0395-500g | |
Magnetic stand | WPI | M1 | |
Magnifying desk lamp | Brightech | LightView Pro Flex 2 | |
Metal Spatula | N/A | N/A | |
MgCl2 . 6H2O | Fisher | BP214-500 | |
Micromanipulator (Right handed) | WPI | M3301R | |
Micromanipulator with digital display | KOPF | 1940 | |
Na2HPO4 . 7H2O | Fisher | S373-500 | |
NaCl | Sigma | S7653-5Kg | |
NaH2PO4 . H2O | Fisher | S369-500 | |
Neosporin | Johnson & Johnson | N/A | Apply topical oinment to prevent infection |
Parafilm | Bemis | DM-999 | |
Rimadyl (Carprofen) 50mg/ml | Zoetis | N/A | 5 mg/kg, subcutaneous, for analgesia |
Scalpel | N/A | N/A | |
Stereotaxic allignment system | KOPF | 1900 | |
Sterile 27 gauge needle | BD | 305109 | |
Sterile cotton tip applicators (Solon) | AMD Medicom | 56200 | |
Sterile nylon sutures (5.0) | Oasis | MV-661 | Use non-absorable suture for closing the wound |
Sterile sharp scissors | N/A | N/A | |
Sterile surgical blades | VWR | 55411-050 | |
Surgical gloves (Nitrile) | Ammex | N/A | Change gloves if there is suspision of contamination |
Tray | N/A | N/A |