Effects of Type 1 Diabetes on the Mechanoreflex in Rats

Mechanical allodynia is present as early as four days post streptozotocin (STZ) injection in type 1 diabetic (T1DM) rats. This is thought to occur through mechanisms affecting the same thin fiber afferents that evoke the mechanoreflex. PURPOSE: In this study, we attempted to determine the effects of T1DM on the mechanoreflex. METHODS: We injected (i.p.) 50 mg/kg of Streptozotocin (STZ) or the vehicle (CTL) in either sex Sprague Dawley rat and waited 1 week (STZ: BW=258±31 g, glucose=448±88 mg/dL, HbA1C=6.4±1.0%; CTL: BW=318±54 g, glucose=175±48 mg/dL, HbA1C=4.3±0.2%). On the day of experiment, the right jugular vein and both carotid arteries were cannulated to inject fluids and to measure blood pressure and heart rate, respectively. The rat was placed in a Kopf stereotaxic frame and spinal unit to perform a precollicular decerebration that allowed for termination of anesthesia. The musculature of the left hindlimb was exposed and the Achilles tendon was attached to a force transducer. The tendon was then stretched for 30 seconds using a rack and pinion and the pressor and cardioaccelerator responses were measured. RESULTS: We found that the pressor (STZ: ΔMAP=42.11±8 mmHg, n=9; CTL: ΔMAP=18.67±4 mmHg, n=6; p=0.02) but not the cardioaccelerator (STZ: ΔHR=13.67±3 bpm, n=9; CTL: ΔHR=9.67±2 bpm, n=6; p=0.22) responses to tendon stretch were exaggerated 1 week after injecting STZ. Both diabetic and control rats developed similar tensions with tendon stretch. CONCLUSION: We conclude that the mechanoreflex is augmented in T1DM rats. Further studies are needed to identify the mechanisms involved in this augmentation

Exercise Pressor Reflex in Type 1 Diabetic Rats is Not Different Between Sexes

Diabetes is a major risk factor for cardiovascular disease and is associated with complications such as autonomic and peripheral neuropathy. The pathophysiology and prevalence of cardiovascular disease differ between sexes. Diabetic patients have an exaggerated pressor and sympathetic response to exercise compared to healthy individuals, which may increase the risk of myocardial infarction and stroke during physical activity. PURPOSE: The purpose of this study was to determine whether the cardioaccelerator and pressor responses to static contraction and tendon stretch differ between sexes in type 1 diabetic (T1DM) rats. METHODS: We injected 50 mg/kg Streptozotocin (STZ) or the vehicle (CTL) i.p in fasted female and male Sprague Dawley rats and waited 10.5±2.5 days (female CTL: BW=274±5 g, glucose=195±12 mg/dL, HbA1C=4.4±0.07%; male CTL: BW=384±4 g, glucose=189±3 mg/dL, HbA1C=4.7±0.1%; female STZ: BW=259±9 g, glucose=485±20 mg/dL, HbA1C=7.3±0.4%; male STZ: BW=294±10 g, glucose=458±17 mg/dL, HbA1C=9.6±0.6%) before performing experiments. All experiments were performed on unanaesthetized, decerebrated rats. We either statically contracted the hind limb muscles or stretched the Achilles tendon for 30 s and measured changes in mean arterial pressure (MAP) and heart rate (HR). RESULTS: We found that the pressor (female CTL: ΔMAP=15±1 mmHg, n=8; male CTL: ΔMAP=16±1 mmHg, n=9; female STZ: ΔMAP=29±6 mmHg, n=8; male STZ: ΔMAP=25±3 mmHg, n=9, p=0.62) and cardioaccelerator (female CTL: ΔHR=17±2 bpm, n=8; male CTL: ΔHR=12±1 bpm, n=9; female STZ: ΔHR=13±5 bpm, n=8; male STZ: ΔHR=24±5 bpm, n=9, p=0.051) responses to static contraction were not significantly different between sexes in T1DM rats. Likewise, the pressor (female CTL: ΔMAP=21±6 mmHg, n=8; male CTL: ΔMAP=33±2 mmHg, n=9; female STZ: ΔMAP=37±8 mmHg, n=10; male STZ: ΔMAP=31±5 mmHg, n=12, p=0.11) and cardioaccelerator (female CTL: ΔHR=9±2 bpm, n=8; male CTL: ΔHR=12±1 bpm, n=9; female STZ: ΔHR=12±4 bpm, n=10; male STZ: ΔHR=14±3 bpm, n=12, p=0.33) responses to tendon stretch were not different between sexes in T1DM rats. The developed tensions from contraction or tendon stretch were similar within each comparison (p\u3e0.05). CONCLUSION: We conclude that the pressor and cardioaccelerator responses to static contraction and tendon stretch are not different between female and male T1DM rats

Augmented Mechanoreflex in Type 2 Diabetic Rats: Piezo Channels, an Important Part of the Puzzle?

Type 2 diabetics (T2DM) have an abnormal cardiovascular response to exercise. The exercise pressor reflex, which is evoked by metabolic and mechanical stimuli arising from the contracting muscle, is a critical cardiovascular regulatory mechanism during exercise and is exaggerated in hypertension, heart failure, and peripheral artery disease. A recent study found that T2DM patients have an augmented metaboreflex. However, whether the mechanoreflex is also augmented in T2DM is not known. PURPOSE: The purpose of the study was to test whether the mechanoreflex is exaggerated in T2DM. Furthermore, we tested the contribution of mechano-gated Piezo 1 and 2 channels to the mechanoreflex in T2DM. METHODS: In unanaesthetized, decerebrated rats we stretched the Achilles tendon for 30 s and measured changes in mean arterial pressure (MAP) and heart rate (HR) in 12 mo old male T2DM rats (BW=546±26 g, glucose=549±28 mg/dl, HbA1c=12.82±0.18%) and healthy male controls (CTL: BW=453±22 g, glucose=229±31 mg/dl, HbA1c=4.6±0.1%). To test the contribution of Piezo channels, we injected GsMTx-4 (10 mg), a known antagonist of Piezo 1 and 2 channels, into the arterial supply of the hindlimb and repeated the stretch maneuver. RESULTS: We found that the pressor (T2DM: ΔMAP=69±6 mmHg, n=5; CTL: ΔMAP=13±2 mmHg, n=5) and cardioaccelerator (T2DM: ΔHR=28±4 bpm, n=5; CTL: ΔHR=5±3 bpm, n=5) responses to tendon stretch were significantly greater in T2DM rats compared to CTL; pCONCLUSION: We conclude that T2DM significantly exaggerates the pressor and cardioaccelerator response to mechanoreflex activation and that Piezo channels play a significant role in evoking the mechanoreflex in T2DM rats

Detection of P2X3 in DRG Using an Automated Approach to Immunoblotting, Jess

Exaggerated cardiovascular (CV) responses to exercise can lead to adverse CV events. Previous studies have reported that P2X3 receptors, found on the peripheral endings of afferents, contribute to an exaggerated exercise pressor reflex in individuals with CV-related diseases. One way to investigate the role played by these receptors in CV pathophysiology is through immunoblotting. The Jess (Protein Simple) provides an automated option for the protein separation and immunoblotting of the traditional Western Blot and allows for total protein staining, an improvement over the use of loading controls to normalize for sample loading variability. PURPOSE: The purpose of this study was to develop a protocol, using the Jess, for quantifying P2X3 receptor protein expression in the L4 and L5 dorsal root ganglia (DRG) of healthy and type 1 diabetic rats. METHODS: Streptozotocin (STZ), 50 mg/kg, or a vehicle (CTL) was injected i.p into fasted Sprague Dawley rats (n=7 each group). After a minimum of 3 weeks, L4 and L5 DRG were excised, immediately placed in HBSS, and then stored at -80°C until subsequent analyses. For quantification, samples were lysed and protein was isolated (Macherey-Nagel) and then quantified (Qubit protein assay kit). For an initial Optimization Run a single test sample lysate was used; different protein concentrations (0.1 - 1.4 mg/ml) were tested against multiple Anti-P2X3 (Novus Biologicals) dilutions (1:25 – 1:250). Sample lysates (3 μl) and required reagents were loaded into a microplate as per manufacturer’s instructions and then the microplate and capillaries were loaded into the Jess. Over 3 hours; protein separation, antibody incubations, washes, and detection were all performed automatically within the Jess. The data from that run provided the optimal protein concentration and antibody dilution that were then used for the Sample Run, which involved running the CTL and STZ sample lysates. For the Sample Run, protein normalization reagent was added to the microplate in order to normalize for sample loading variability using total protein staining. RESULTS: Optimization Run - A protein concentration of 1.4 mg/ml and a dilution of 1:250 for the P2X3 antibody were found to be optimal. This determination was based on the combination of a low background signal from the antibody and a detectable target protein signal. Sample Run - We found that P2X3 receptor protein expression decreased in STZ rats compared to CTL rats (0.82 ± 0.09 vs 1.00 ± 0.19; n=7 both groups, p=0.03). CONCLUSION: Using the Jess, an automated protocol was developed that detected differences in P2X3 receptor protein expression in rats with and without diabetes. Advantages over the traditional Western Blot include: a run time of 3 hours, reduced user-associated variability thru automation, and the capability of using total protein staining to normalize for sample loading variability. Technological advances such as the Jess are a step towards addressing current rigor and reproducibility concerns

Quantification Method of P2X3 Receptors in Rat DRG Neurons: Western Blotting

Skeletal muscle contractions are known to evoke pressor and cardioaccelerator responses in part by stimulating P2X3 receptors found on the peripheral endings of afferents. In diabetic patients, this pressor response is exaggerated. What is currently not known is whether P2X3 receptors play a role in evoking this exaggerated response. PURPOSE: The purpose of this project was to quantify P2X3 receptors in the L4 and L5 dorsal root ganglia (DRG) neurons in both healthy and type 1 diabetic rats using western blot analysis. METHODS: We injected 50 mg/kg streptozotocin (STZ) or the vehicle (CTL) i.p in fasted female and male Sprague Dawley rats and then waited at least 7 days for the rats to become diabetic. We then performed a laminectomy in the anesthetized rats to expose the spinal cord and roots. Using a dissecting microscope, we removed the L4 and L5 DRG from the spinal column. The DRG are the cell bodies of the peripheral afferents found in the hindlimb musculature. The DRG were placed in HBSS (is this buffer?) and stored at -80°C until analysis. For quantification, samples were lysed and proteins were isolated using the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Bethlehem, PA, USA). A Qubit 3.0 Fluorometer was used to quantify the protein concentration of each sample so that equal protein concentrations could then be loaded onto a Bolt Bis-Tris (4-12%) gel. Following electrophoresis, the proteins were transferred to a membrane before being probed with a rabbit polyclonal P2X3 antibody (Alomone Labs), followed by an anti-rabbit secondary antibody conjugated to alkaline phosphatase (Life Technologies). The membrane was then exposed using a ChemiDoc XRS and the results analyzed using BioRad’s Quantity One imaging software. RESULTS: We were able to detect P2X3 receptor proteins. When compared with a molecular weight ladder, P2X3 receptor proteins were 54kDa, which is similar to the molecular weight of P2X3 receptors quantified in other studies. CONCLUSION: This method of quantifying P2X3 receptors in DRG neurons allows for a comparison between non-diabetic and diabetic rats. Further analyses are required to determine whether the quantity of P2X3 receptors in L4 and L5 DRG neurons is different in diabetic rats compared to non-diabetic rats

Acute Effect of Hyperglycemia on the Mechanoreflex and Metaboreflex

Recent studies in both humans and rodents have shown that the mechanoreflex and metaboreflex are exaggerated in type 2 diabetes mellitus (T2DM). Hyperglycemia is a main characteristic of T2DM and is known to cause damage to both cardiovascular and nervous system structures. However, the acute effect of the presence of hyperglycemia on the mechanoreflex and metaboreflex are not known. PURPOSE: To determine the acute effect of hyperglycemia on the mechanoreflex and metaboreflex. METHODS: Experiments were conducted after an overnight fast in unanesthetized, decerebrated healthy male and female Sprague-Dawley rats. The mechanoreflex was evoked by stretching the Achilles tendon for 30 s whereas the metaboreflex was evoked by locally injecting lactic acid (0.2ml, 24mM) into the hindlimb. Time and dosage for glucose infusion were selected based on a preliminary study that showed infusing 250 mg/ml of glucose solution for 15 min into the hindlimb circulation, with blood flow to and from the hindlimb restricted, would elevate local blood glucose concentration to the same degree as that seen in T2DM rats with an exaggerated exercise pressor reflex. To elicit an acute local hyperglycemic environment, while preventing an endogenous insulin response, somatostatin (3.9 ug/100 ul) was infused systemically and simultaneously with the local glucose infusion. Changes in mean arterial pressure (ΔMAP) and heart rate (ΔHR) in response to tendon stretch and lactic acid injection were measured and compared before and after infusion. RESULTS: We found that the peak pressor and cardioaccelerator responses to tendon stretch were not significantly affected by hyperglycemia (ΔMAP before: 12 ± 2 mmHg, after: 12 ± 3 mmHg, n=6, p\u3e0.05; ΔHR before: 10 ± 3 bpm; after: 10 ± 3 bpm, n=6, p\u3e0.05). Likewise, the pressor and cardioaccelerator responses to lactic acid were not significantly affected by hyperglycemia (ΔMAP before: 13 ± 2 mmHg, after: 16 ± 3 mmHg, n=10, p\u3e0.05; ΔHR before: 10 ± 2 bpm, after: 12 ± 5 bpm, n=10, p\u3e0.05). CONCLUSION: The acute presence of hyperglycemia in the local circulation of the hindlimb likely does not contribute to the exaggerated mechanoreflex or metaboreflex

Trabecular and Cortical Bone and Ossified Vessel Alterations in Rat Tibiae with the Onset and Progression of Type 2 Diabetes Mellitus in a Novel, Transgenic Rat Model

Type 2 Diabetes Mellitus (T2DM) is a metabolic disorder of systemic complications including increased fracture risk and microvascular pathology, suggesting a potential link between the two. PURPOSE: We determined how the onset and progression of T2DM affected bone and marrow vasculature in the University of California Davis T2DM transgenic rat model. METHODS: Forty-eight male T2DM rats were divided accordingly: pre-diabetes (12wks), diabetes onset (14wks), early-stage diabetes (20wks; 4wks post-onset), and late-stage diabetes (22wks; 12wks post-onset) matched with four healthy control (CTL; Sprague Dawley) groups. Body mass(g), HbA1c(%), and fasted blood glucose(g/dL) were measured at sacrifice. Tibiae were scanned via µCT (15µm) to assess trabecular volume-to-total volume ratio (BV/TV, %), trabecular thickness (Tb.Th, µm), trabecular number (Tb.N, /mm), trabecular separation (Tb.Sp µm), and density (mgHA/ccm) in the proximal metaphysis. Cortical thickness (Ct.Th, µm) and density (mmHg/ccm) were measured at the mid-shaft, and cortical porosity (%) was calculated (1-Ct.BV/TV). Ossified vessel volume (OsVV, %), ossified vessel thickness (OsV.Th, µm), and OsV density (mgHA/ccm) were analyzed in the diaphyseal marrow, representing conversion of blood vessels into bone-like tissue. A General Linear Model determined significance at p\u3c0.05, a priori. RESULTS: Body mass (455-622g vs. 342-435g) and HbA1c (5-12% vs. 5%) was higher (p\u3c0.05) in the T2DM vs. CTL groups, respectively. Blood glucose rose (p\u3c0.05) in early- (113±9g/dL vs. 71±7g/dL) and late- (244±10g/dL vs. 68±2g/dL) stage diabetes vs. CTL. Trabecular BV/TV was lower (p\u3c0.05) in pre- (4±1% vs. 9±2%) and late-stage (5±2% vs. 8±2%) diabetes vs. CTL, from reduced (p\u3c0.05) Tb.N in pre- (2.5±0.1/mm vs. CTL, 3.8±0.2/mm) and late-stage (2.1±0.3/mm vs. CTL, 2.6±0.4/mm), and reduced (p\u3c0.05) Tb.Th in late-stage (56±3µm vs. CTL, 67±4µm) diabetes. Trabecular separation increased (p\u3c0.05) in pre-(407±23µm vs. CTL, 263±15µm) and late-stage (482±85µm vs. CTL, 406±85µm). Trabecular density and Ct.Th, density, and porosity did not differ. OsVV was lower (p\u3c0.05) in early-stage diabetes (1.7±0.2% vs. CTL, 4.7±1.5%), OsV.Th was higher (p\u3c0.05) in pre-(69±14µm vs. CTL, 56±13µm) and late-stage (80±10µm vs. CTL, 59±13µm) diabetes, and OsV density was higher (p\u3c0.05) in late-stage diabetes (918±17mgHA/ccm vs. CTL, 891±31mgHA /ccm).CONCLUSION: T2DM developed in the transgenic rat model (i.e., increases in HbA1c, and blood glucose). Cortical bone parameters were not altered. Trabecular bone declined in pre- and late-stage diabetes, via reduced trabecular number and thickness. Ossified vessels were thicker at these stages. Thus, the observed trabecular bone and vascular pathologies coincided in the tibia with the onset and progress of T2DM

Using HbA1c to Diagnose Diabetes in the UC, Davis-Type 2 Diabetes Mellitus Rat Model

With disease progression, type 2 diabetes mellitus (T2DM) leads to debilitating complications arising from damage to nerves and blood vessels. Importantly, investigations focusing on T2DM progression have the capacity to distinguish individuals at greater risk for these severe complications through the identification of predictive biomarkers. Hence, the accurate diagnosis of T2DM is critical to such investigations. UC Davis (UCD) T2DM rats are born without diabetes and develop the disease over time with a similar pathophysiology to that in humans. This unique rat model allows researchers to investigate predictive biomarkers linked to the progression of T2D; however, such investigations require an accurate diagnosis of T2DM onset. PURPOSE: To determine the most accurate measure to diagnose T2DM using UCD-T2DM rats. METHODS: 10 male UCD-T2DM rats were used in this study. Glucose and HbA1c were measured weekly from the tail beginning at 16 wks of age (before onset) and continuing until 25 wks of age (all rats had become diabetic). These measures were taken under both fasted (8 hrs) and random conditions as well as in the morning (AM) and afternoon (PM). A two-way repeated measures ANOVA was run with condition [fasted (FG) vs random (RG)] and time (AM vs PM) as factors, followed by Holm Sidak post hoc analyses. In addition, growth curves were fit to the data for all rats to estimate the trajectories of RG and HbA1c. RESULTS: We found that RG was more variable compared to FG (FG: 116±46 vs RG: 216±94 mg/dL; n=10). However, HbA1c was stable across both conditions (fasted HbA1c: 6.0±0.8 vs random HbA1c: 6.0±1.0%; n=10). In addition, both FG and RG morning levels were significantly lower compared to afternoon (FG AM: 99±6 vs FG PM: 133±19 mg/dL; n=10; p0.05). In addition, the location on the growth curve where RG crossed 200 mg/dL (currently the most common diagnostic criteria used) corresponded to a HbA1c of 5.6%. CONCLUSION: A HbA1c of 5.6% may provide a more accurate measure to diagnose the onset of diabetes in the UCD-T2DM rat model

Bubbles navigating through networks of microchannels

This paper describes the behavior of bubbles suspended in a carrier liquid and moving within microfluidic networks of different connectivities. A single-phase continuum fluid, when flowing in a network of channels, partitions itself among all possible paths connecting the inlet and outlet. The flow rates along different paths are determined by the interaction between the fluid and the global structure of the network. That is, the distribution of flows depends on the fluidic resistances of all channels of the network. The movement of bubbles of gas, or droplets of liquid, suspended in a liquid can be quite different from the movement of a single-phase liquid, especially when they have sizes slightly larger than the channels, so that the bubbles (or droplets) contribute to the fluidic resistance of a channel when they are transiting it. This paper examines bubbles in this size range; in the size range examined, the bubbles are discrete and do not divide at junctions. As a consequence, a single bubble traverses only one of the possible paths through the network, and makes a sequence of binary choices (“left” or “right”) at each branching intersection it encounters. We designed networks so that, at each junction, a bubble enters the channel into which the volumetric flow rate of the carrier liquid is highest. When there is only a single bubble inside a network at a time, the path taken by the bubble is, counter-intuitively, not necessarily the shortest or the fastest connecting the inlet and outlet. When a small number of bubbles move simultaneously through a network, they interact with one another by modifying fluidic resistances and flows in a time dependent manner; such groups of bubbles show very complex behaviors. When a large number of bubbles (sufficiently large that the volume of the bubbles occupies a significant fraction of the volume of the network) flow simultaneously through a network, however, the collective behavior of bubbles—the fluxes of bubbles through different paths of the network—can resemble the distribution of flows of a single-phase fluid.Chemistry and Chemical Biolog

Obesity Contributes to an Attenuated Spontaneous Baroreflex Sensitivity in UCD-Type 2 Diabetic Rats

Previous studies suggest impaired baroreflex function in individuals with type 2 diabetes (T2D), which is critically important since it leads to an increased risk for adverse cardiovascular events. Currently, the underlying mechanisms remain poorly understood. The baroreflex, essential for maintaining blood pressure homeostasis, can also be influenced by several risk factors, one of which is obesity. Obesity has been shown to markedly decrease baroreflex sensitivity (BRS) in non-diabetic individuals, and given that the majority of T2D patients are obese, it is likely that impairment in baroreflex function in T2D is mainly driven by obesity. PURPOSE: To investigate the effects of obesity on baroreflex function in T2D rats at different phases of the disease. We hypothesized that BRS would be attenuated in T2D rats, and this would be associated with increased adiposity. METHODS: Experiments were performed on male University of California Davis (UCD)-T2D rats assigned to four experimental groups (n=6 in each group): prediabetic (PD), diabetes-onset (DO), 4 weeks after onset [recent-onset (RO)], and 12 weeks after onset [late-onset (LO)]. Age-matched healthy Sprague-Dawley rats were assigned to the same experimental groups as controls (n=6 in each). Rats were anesthetized and blood pressure was directly measured for 5 min. Hemodynamic variables were obtained on a beat-to-beat basis and spontaneous BRS was assessed using the sequence technique. Dual-energy X-ray absorptiometry (DEXA) was used to assess body composition and visceral fat was determined by identifying an abdominal region of interest. Data are presented as mean ± SD. RESULTS: Spontaneous BRS was significantly lower in T2D compared to control rats at DO (3.7 ± 3.2 ms/mmHg vs 16.1 ± 8.4 ms/mmHg; P=0.01). However, this difference was abolished by LO (13.4 ± 8.1 ms/mmHg vs 9.2 ± 6.0 ms/mmHg; P=0.16). T2D rats had the highest level of adiposity during the RO phase but it significantly decreased by LO (PD: 136 ± 14 g; DO: 175 ± 24 g; RO: 207 ± 44 g; LO: 163 ± 45 g; P=0.03). In addition, T2D rats had greater visceral fat compared to control rats regardless of the disease phase (P\u3c0.01). CONCLUSION: These findings suggest that obesity may contribute to an attenuated spontaneous BRS in T2D rats and suggests a link between metabolic and autonomic dysfunction in T2D