The Effects of Acute Blood Loss for Diagnostic Bloodwork and Fluid Replacement in Clinically Ill Mice

Abstract

Despite the great value of diagnostic bloodwork for identifying disease in animals, the volume of blood required for these analyses limits its use in laboratory mice, particularly when they are clinically ill. We sought to determine the effects of acute blood loss (ABL) following blood collection for diagnostic bloodwork in healthy mice compared with streptozotocin-induced diabetic and dextran sulfate sodium (DSS)-treated dehydrated mice. ABL caused several mild changes in the control mice, with significant decreases in body weight, temperature, and activity in both experimental groups; increased dehydration and azotemia in the DSS-treated mice; and a significant drop in the blood pressure of the diabetic mice. To determine whether these negative outcomes could be ameliorated, we treated mice with intraperitoneal lactated Ringers solution either immediately after or 30 min before ABL. Notably, preABL administration of fluids helped prevent the worsening of the dehydration and azotemia in the DSS-treated mice and the changes in blood pressure in the diabetic mice. However, fluid administration provided no benefit in control of blood pressure when administered after ABL in the diabetic mice. Furthermore, fluid therapy did not prevent ABL-induced drops in body weight and activity. Although one mouse not receiving fluid therapy became moribund at the 24-h time point, no animals died during the 24-h study. This investigation demonstrates that blood for diagnostic bloodwork can be collected safely from clinically ill mice and that preemptive fluid therapy mitigates some of the negative changes associated with this blood loss.

One of the most valuable clinical tools available to veterinarians is diagnostic bloodwork, because routine CBC and serum chemistry profiles provide important information about many body systems and can direct subsequent diagnostic testing and therapeutic strategies. Unfortunately, these important screening tests are performed only rarely in mice, due to their small size and the volume of blood required. These difficulties are further compounded when working with sick mice, which may be negatively, if not critically, affected through the loss of a significant percentage of their blood volume when collected for diagnostic assessments.

Performing routine CBC and serum chemistry analyses in mice requires approximately 10% to 15% of an adult mouse's total blood volume. The physiologic response to acute blood loss (ABL) has been well characterized in healthy rodents. In healthy animals, the loss of less than 15% of the blood volume initiates compensatory mechanisms, including increased heart rate, arterial and venous contraction, and hormonal changes that maintain cardiac output, venous return, and arterial blood pressure (BP) and collectively prevent overt adverse physiologic changes.^29,33 Therefore the 10% to 15% blood loss from healthy mice for CBC and serum chemistry analysis likely will be well tolerated. However, as the percentage of total blood volume lost increases, these compensatory mechanisms become insufficient, and tissue hypoxia, hypercapnia, and acidosis that lead to cell injury and death can rapidly ensue. Ultimately, large volumes of blood loss compromise blood flow to the heart and brain and may be fatal.²⁹

Although the physiologic responses to ABL in healthy rodents have been well described, ABL-induced effects in clinically ill or compromised mice are not well characterized. Because the compensatory mechanisms described earlier may have already been activated in dehydrated mice, their ability to maintain cardiac output and BP in the face of ABL may be limited, resulting in tissue hypoxia, hypercapnia, and acidosis with even small volumes of blood loss. Furthermore, animals with metabolic disease may be unable to initiate these compensatory mechanisms effectively; for example, untreated diabetes mellitus (DM) significantly compromises the body's ability to compensate for ABL.⁴ Specifically, in addition to changes in the resting heart rate and BP, DM rats with ABL exhibited a significant decrease in BP at 12% blood loss and a decrease in heart rate at 18% blood loss. In the control rats, the BP decrease did not occur until 18% blood loss, and the heart rate did not decrease.⁴⁰ A second example of the importance of the animal's condition at the time of a blood loss was demonstrated in a study in which 2 blood losses of 2% to 20%, separated by just 30 min, had a more profound negative effect on the immune system than did a single 50% blood loss.⁵³

The goal of the current study was to characterize the effects of 15% blood loss on basic health, diagnostic bloodwork, and cardiovascular parameters in 2 common conditions, dehydration and DM, that may compromise a mouse's ability to compensate for ABL. Dehydration was secondary to DSS treatment to induce inflammatory bowel disease, which in combination with ABL, creates a severe risk of hypovolemic shock. DM was induced by treatment with streptozotocin, which has well-known effects on metabolism^8,18,26 and is frequently associated with cardiovascular autonomic neuropathy. All of these factors may significantly impair the mouse's ability to compensate after ABL.⁴

Finally, the standard treatment for ABL across species is volume replacement with fluids—crystalloid, colloids, or whole blood—to maintain blood pressure and cardiac output until the source of the blood loss is controlled.^37,48 Experimental studies of healthy dogs and NHP have shown that euvolemic animals can maintain adequate tissue oxygen delivery at Hct values below 10%.^7,42 Therefore, an additional objective of the current study was to test the ability of intraperitoneal lactated Ringers solution to maintain adequate plasma volume and support cardiac output and tissue oxygenation, thereby ameliorating the negative effects of ABL in these compromised mice. We chose intraperitoneal administration of fluids for these studies because of its ease of administration to laboratory mice and because it is an effective alternative to intravenous therapy under various conditions.^2,51

Materials and Methods

Young adult (6 to 10 wk) male and female C57BL/6J mice (Mus musculus; Jackson Laboratory, Bar Harbor, ME) were maintained on a 12:12-h light:dark cycle and housed in polycarbonate cages with corncob bedding, with free access to autoclaved food (Lab Diet 5010, Animal Specialties and Provisions, Quakertown, PA) and water. Before the start of the study, the mice were allowed at least 1 wk to acclimate to the housing facility and cage environment. Sentinel mice were tested routinely and found to be free from pinworms by cecal exam and were negative for antibodies to tested pathogens including mouse hepatitis virus, mouse parvoviruses, rotavirus, ectromelia virus, Sendai virus, pneumonia virus of mice, Theiler murine encephalomyelitis virus, reovirus, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus, and polyomavirus. All aspects of the current investigation were approved by the University of Pennsylvania IACUC.

The 3 groups of mice included healthy control mice, streptozotocin-induced DM mice, and mice dehydrated due to inflammatory bowel disease induced by adding DSS to the drinking water. DM was induced by treatment with streptozotocin (50 mg/kg daily for 5 consecutive days) at the vendor (Jackson Laboratory, Bar Harbor, ME) and was confirmed by blood glucose values that exceeded 300 mg/dL at multiple time points.³⁴ The initial glucose measurements were performed by the vendor, approximately 2 wk before the actual experiments, and subsequent evaluation of the blood glucose was performed as part of the current study. Because of the variable response of female mice to streptozotocin treatment, only male mice were used in this group.^8,25,26 Inflammatory bowel disease was induced by the administration of 5% DSS for 3 d. Both male and female mice were used as control and DSS-treated mice in experiment 1. In experiment 2, only male mice were used to prevent any confounding effects of sex, given that only male diabetic mice were tested.

One week before the experiments were performed, conscious mice were implanted subcutaneously on the dorsum between the scapulae with individual transponders (model IPTT-300, BMDS, Seaford, DE) that transmit body temperature to a scanning device system (model DAS-6007, BMDS). All microchip transponders were inserted as directed by the manufacturer and successfully provided data throughout the course of the experiments; manufacturer instructions did not indicate that calibration, beyond initial factory settings, was necessary. In addition, the mice were acclimated to the individual restraint tubes required for BP monitoring. The mice underwent 4 sessions, beginning with 2 min of restraint and increasing to 10 min of restraint, similar to the time of restraint during the actual BP measurements.

Experiment 1.

In experiment 1, 22 control (11 male and 11 female), 19 male DM, and 20 DSS (14 male and 6 female) mice were used. On the day of the experiment, mice were scanned to obtain the body temperature and then weighed. The mice then were placed into a novel clean cage with fresh bedding and were videotaped (Coolpix L24, Nikon, Tokyo, Japan) for 5 min to assess their activity when placed in a new environment. The videos were analyzed by using VideoPad Video Editor Software (NCH Software, Greenwood Village, CO), which imposed a grid over the video image to divide the cage into 5 equally sized horizontal sections. Activity was assessed by monitoring the movement of a point between the ears as the mouse moved across the grid.

Immediately after the activity monitoring, the heart rate was determined by using electrocardiography (ECGenie and eMouse 11 Analysis Software, Mouse Specifics, Quincy, MS). Mice were acclimated to the ECG platform for at least 3 min before recording the heart rate. Directly after heart rate recordings, the systolic, diastolic, and mean BP were measured by using plethysmography of the tail (CODA Mouse and Rat Tail-Cuff Blood Pressure System, KENT Scientific CO., Connecticut). Mice were placed individually in a restrainer that allowed free access to the tail. The mice restrainers were placed on a warming tray for approximately 5 min to ensure adequate blood flow to the tail. Ten measurements were recorded over 5 min, and valid readings were averaged to determine the values for systolic, diastolic, and mean BP.

For blood collection, mice were anesthetized with isoflurane in an induction chamber, where they were exposed to 3% isoflurane until they lost their righting reflex. Mice then were maintained within the chamber for 3 min at 2.25% isoflurane. This protocol provided sufficient anesthesia to collect approximately 250 µL of blood by using retroorbital vascular access after removal from the chamber. We opted for retroorbital bleeding over other sampling routes because of greater control of the volume of blood collected. The total volume of blood loss in the mice was recorded as the volume of blood collected in the centrifuge tube. A small percentage of mice lost additional blood after removal of the capillary tube; this amount was estimated and added to the volume of blood collected for the total volume of blood. Control mice, which weighed more than the mice in the 2 experimental groups, had a slightly greater volume of blood collected to keep the percentage of blood loss equal among the 3 groups (Table 1). The percentage of blood loss was calculated according to a blood volume equal to 7% of the animal's body weight, independent of the experimental group.¹⁰ This estimate was confirmed in the dehydrated, DSS-treated mice in light of the assumption that the total volume of RBC did not change over the 3 d of DSS treatment (no frank blood was noted in the feces), and the change in Hct was due solely to the change in the plasma volume. The mice were allowed to recover from anesthesia and were returned to their home cages. CBC and serum chemistry analyses were performed on each collected sample; the CBC consisted of a manual differential cell count and analysis (Vet ABC Hematology Analyzer, Scil Animal Care, Gurnee, IL), and the chemistry analysis was performed on a Vitos 250 Chemistry Analyzer (Ortho Clinical Diagnostics, Auckland, New Zealand).

Table 1.

Volume of blood and percentage of total blood volume collected from each group of mice

			Experiment 2
	Experiment 1		Fluids administered before ABL		Fluids administered after ABL
Mice	Volume of blood (µL)	Percentage (%) of total blood volume	Volume of blood (µL)	Percentage (%) of total blood volume	Volume of blood (µL)	Percentage (%) of total blood volume
Control	308 ± 18a	16.3 ± 1.0	261 ± 26	15.6 ± 0.5	311 ± 10a	16.8 ± 0.8
DM	242 ± 10	15.3 ± 0.7	256 ± 7	16.9 ± 0.7	250 ± 12	18.2 ± 1.1
DSS-treated	235 ± 10	15.7 ± 0.8	264 ± 13	17.6 ± 0.9	260 ± 6	17.4 ± 0.5

Data are given as mean ± SE.

^adenotes a significant difference from the other blood volumes collected within the experiment

Mice were reevaluated at either 4 or 24 h after ABL. To maintain the level of pathology observed before ABL in mice with inflammatory bowel disease, the concentration of DSS supplied in the drinking water differed between the 2 time points. Specifically, the mice reevaluated at the 4-h time point remained on drinking water that contained 5% DSS, whereas mice reevaluated at the 24-h time point were switched to 1.5% DSS in water, given that pilot experiments demonstrated excessive weight loss after the initial ABL when the mice were maintained on 5% DSS for an additional 24 h. Notably, C57Bl/6J mice are extremely sensitive to DSS.^28,31,38 The only difference between the baseline and the second time point of testing was that mice were euthanized after the second time point, thus allowing collection of the maximal blood volume while the mice were under deep anesthesia.

Experiment 2.

In experiment 2, 15 control, 21 DM, and 32 DSS male mice were used. The testing procedures were the same as in experiment 1, except that the mice received 1.5 mL of intraperitoneal lactated Ringers solution, warmed to body temperature, at 1 of 2 time points relative to the blood collection.¹¹ Briefly, the data for body temperature and weight, activity, heart rate, and blood pressure were collected. One group received the fluids immediately after the blood was collected, while the mice were still anesthetized, and the second group received the fluids 30 min before being anesthetized with isoflurane for blood collection, to determine whether fluid administration had a protective effect in regard to ABL. In both groups, the body temperature, weight, activity, heart rate, and BP data were collected before intraperitoneal fluid administration, meaning that only the measurements for CBC and serum chemistry analysis were influenced by fluid administration. The second time point in the second experiment was 24 h after ABL. An additional group of DSS-treated mice that received fluids immediately after ABL was analyzed at the 4-h time point. As in the first experiment, mice tested at the 4-h time point continued to receive 5% DSS in the drinking water after ABL; mice tested at the 24-h time point received 1.5% DSS in the drinking water after ABL.

Statistical analysis.

Experiment 1.

Differences between the 3 groups at baseline, before ABL, were analyzed for male mice only (because no female diabetic mice included in the study) by one-way ANOVA for normally distributed data and by Kruskal–Wallis analysis for data that were not normally distributed. Differences between male and female mice in the control and DSS-treated groups were analyzed by t tests for normally distributed data and by the Mann–Whitney rank-sum test for data not normally distributed. A flow chart of the statistical analysis of the effects of ABL and the dependent variables is provided in Figure 1. Data from male and female mice were pooled for variables that had no sex-associated differences. Data that showed normal distribution were analyzed by repeated-measures, 2-way ANOVA (main effects were experimental group and time point). For data that showed no sex-associated differences and were not normally distributed, the 3 experimental groups were analyzed individually by one-way repeated-measures ANOVA (main effect was time point). When the data were not normally distributed at this point, a Friedman repeated-measures ANOVA on ranks was performed.

Figure 1.

Flow chart of statistical analysis of the effects of ABL for experiment 1. When no sex-associated differences were detected, data from male and female mice were pooled together. When sex-associated differences emerged, the data were analyzed separately by sex.

When sex-associated differences were detected in either the control or DSS-treated mice, the data were analyzed by sex in the 3 experimental groups. In the experimental groups in which sex-associated differences were detected, the comparison between the baseline and 4-h data was performed by using the female mice and that between the baseline and 24-h data was done by using the male mice in a one-way, repeated-measures ANOVA for normally distributed data or a Friedman repeated-measures ANOVA on ranks for data without normal distribution. In the DM mice or when there was no sex-associated difference in the experimental group, the data from male and female mice were pooled for analysis and analyzed by one-way, repeated-measures ANOVA for normally distributed data or Friedman one-way repeated-measures ANOVA when not normally distributed.

Experiment 2.

A flow chart of the statistical analysis for experiment 2 is provided in Figure 2. The analysis of the dependent variables was performed within each experimental group. The data in the second experiment that showed normal distribution again were analyzed by using 2-way repeated-measures ANOVA for the control and DM mice, with the main effects of time point and time of fluid administration. The DSS-treated mice could not be analyzed in this fashion because of the extra time point (4 h after ABL) for just one fluid-administration time point. The variables for the DSS-treated mice and the variables that did not show normal distribution in the control and DM groups were analyzed according to the time of fluid administration by one-way, repeated-measures ANOVA, with the main effect of time point. If the data did not have a normal distribution at this time point, then Friedman repeated-measures ANOVA on ranks was performed. Last, to determine the effects of fluid administration independent of ABL, a t test was performed to compare the baseline values of the CBC and serum chemistry analyses for the 2 different time points of fluid administration.

Figure 2.

Flow chart of statistical analysis of each independent variable by experimental group in experiment 2.

For both experiments, Tukey post hoc analysis was performed when significant differences were detected by using parametric statistics and Dunn post hoc analysis was used for nonparametric statistics. Significance was set at a P value of less than 0.05, unless the data set was split to achieve a normal distribution, in which case the P value was divided appropriately. All statistical comparisons were performed by using SigmaPlot 12 (Systat Software, San Jose, CA).

Results

The anesthetic protocol used in the collection of blood proved to be highly effective, with no anesthetic deaths associated with the procedure. In the first experiment, a larger (P < 0.05) volume of blood was collected from mice in the control group than in the other 2 groups, but the percentage of blood loss did not differ significantly among the 3 groups (Table 1). In the second experiment, there was a significant (P < 0.05) difference in the blood volume collected between the control and 2 experimental groups in mice receiving fluids after the ABL, but no differences were detected when fluids were administered before ABL (Table 1). Again, no significant differences were detected in the percentage of the total blood volume collected. For the mice that received fluids before ABL, the calculation of blood volume was based on the values obtained before the administration of fluids, because we were unable to reliably determine how much of the fluids had left the peritoneal space and migrated into the vascular space during the 30 min between fluid administration and ABL. Only one mouse in either experiment was identified as becoming moribund during the studies. This mouse was from the DSS-treated group in experiment 1 and was at its predetermined experimental endpoint when it became moribund.

Baseline values of control mice.

The majority of the baseline CBC and plasma chemistry values for the control mice were within the normal range reported by the reference laboratory, except for the total WBC and lymphocytes counts and the plasma albumin and potassium concentrations, all of which were below the reported reference range. Despite the decreased lymphocytes, the mice were lymphocyte-dominant, which is typical for mice.^41,44 The heart rate in the mice was consistent with published reports for the baseline heart rate of C57BL/6 mice.^27,32 The BP measured in our mice was higher than typically is reported in the literature for C57BL/6 mice.²⁰ The body temperatures were typical for C57BL/6 mice. The weights of the control male mice were consistent with the expected values, whereas our female control mice were heavier than expected based on Jackson Laboratory's weigh charts.¹⁹

Baseline differences between groups.

The 3 groups showed several significant differences in the baseline evaluation of the male mice (Table 2). Both the DM and DSS-treated mice were significantly (P < 0.05) smaller than the control mice at the start of the experiment. DSS-treated mice also showed a significant decrease in activity compared with control mice. On physical exam, both groups had marked muscle wasting compared with control mice; the DSS-treated mice also had mild signs of dehydration (skin tenting, ‘dull’ appearance). Compared with the control mice, the DSS-treated mice demonstrated several significant (P < 0.05) changes indicative of dehydration on the serum chemistry profile, including elevations in Hct, BUN, total protein, and albumin. Diagnostic tests on the chemistry profile were often limited due to limited plasma volumes collected during the initial sampling, particularly in the dehydrated, hemoconcentrated DSS-treated mice. No significant changes in creatinine were noted, likely due to the mouse's ability to excrete creatine directly into the urine.³ The cardiovascular changes (P < 0.05) were consistent with dehydration, including an increase in heart rate and decreases in systolic, diastolic, and mean BP.

Table 2.

Baseline differences between experimental groups and sexes

	Control mice		DM mice	DSS mice
	Female	Male	Male	Male	Female
Body weight (g)	28.8 ± 0.5 (n = 10)	26.8 ± 1.0 (n = 11)a	22.8 ± 0.5 (n = 19)b	21.4 ± 0.4 (n = 14)b	22.0 ± 0.6 (n = 6)
Body temperature (°C)	36.7 ± 0.3 (n = 11)d	38.4 ± 0.3 (n = 11)	37.6 ± 0.3 (n = 19)	37.6 ± 0.2 (n = 14)	35.8 ± 0.4 (n = 6)d
Activity (no. of line  breaks in 5 min)	128.2 ± 14.3 (n = 9)	152.0 ± 16.8 (n = 8)a	126.7 ± 8.6 (n = 19)a	82.7 ± 5.3 (n = 9)b	65.0 ± 9.6 (n = 5)
Heart rate (bpm)	714 ± 16 (n = 11)d	744 ± 6 (n = 11)a	716 ± 12 (n = 18)a	787 ± 7 (n = 14)b	758 ± 9 (n = 6)d
Systolic BP (mm Hg)	159.6 ± 2.2 (n = 7)	163.9 ± 4.2 (n = 8)a	161.2 ± 4.1 (n = 18)a	136.7 ± 7.9 (n = 4)b
Diastolic BP (mm Hg)	127.8 ± 2.9 (n = 7)	132.3 ± 4.6 (n = 8)a	128.0 ± 4.2 (n = 18)a	96.2 ± 5.6 (n = 4)b
Mean BP (mm Hg)	138.1 ± 2.5 (n = 7)	142.6 ± 4.4 (n = 8)a	139.9 ± 4.2 (n = 18)a	109.3 ± 6.0 (n = 4)b
Hct (%)	42.1 ± 1.5 (n = 11)	43.5 ± 0.9 (n = 11)a	46.6 ± 0.8 (n = 19)b	57.8 ± 0.6 (n = 13)c	57.7 ± 0.8 (n = 6)
WBC (× 103/µL)	5.0 ± 1.1 (n = 11)	3.3 ± 0.6 (n = 11)	5.0 ± 0.8 (n = 19)	4.8 ± 1.0 (n = 13)	4.2 ± 1.2 (n = 6)
Neutrophils (× 103/µL)	1.2 ± 0.6 (n = 11)	1.5 ± 0.7 (n = 8)	1.0 ± 0.2 (n = 19)	2.5 ± 0.7 (n = 9)	2.0 ± 0.7 (n = 6)
Lymphocytes (× 103/µL)	3.5 ± 0.7 (n = 11)	2.0 ± 0.5 (n = 8)a,b	4.0 ± 0.6 (n = 19)a	1.4 ± 0.5 (n = 9)b	2.1 ± 0.9 (n = 6)
Glucose (mg/dL)	187.4 ± 26.4 (n = 11)	182.3 ± 17.8 (n = 11)a	400.6 ± 33.0 (n = 19)b	133.6 ± 8.1 (n = 11)a	110.2 ± 19.3 (n = 6)
Total protein (mg/dL)	5.3 ± 0.7 (n = 9)	4.7 ± 0.3 (n = 10)a	4.8 ± 0.2 (n = 19)a	6.6 ± 0.2 (n = 11)b	5.6 ± 0.8 (n = 6)
Albumin (g/dL)	2.2 ± 0.3 (n = 9)	1.9 ± 0.1 (n = 10)a	1.8 ± 0.1 (n = 19)a	2.5 ± 0.1 (n = 11)b	1.9 ± 0.2 (n = 6)d
Globulin (g/dL)	3.1 ± 0.3 (n = 9)	2.8 ± 0.2 (n = 10)a	3.0 ± 0.2 (n = 17)a	4.0 ± 0.2 (n = 11)b	4.1 ± 0.6 (n = 5)
BUN (mg/dL)	27.2 ± 2.8 (n = 11)	25.7 ± 2.7 (n = 10)a	23.0 ± 1.4 (n = 19)a	55.2 ± 3.3 (n = 12)b	39.5 ± 5.8 (n = 6)d
Creatinine (mg/dL)	0.3 ± 0.1 (n = 11)	0.2 ± 0.1 (n = 10)a,b	0.3 ± 0.1 (n = 19)a	0.2 ± 0.1 (n = 12)b	0.3 ± 0.1 (n = 6)
ALT (U/L)	95.7 ± 9.2 (n = 11)	103.1 ± 13.3 (n = 10)	113.8 ± 10.0 (n = 18)	103.4 ± 6.0 (n = 9)	83.4 ± 17.6 (n = 6)d
AST (U/L)	138.6 ± 35.5 (n = 11)	128.2 ± 29.0 (n = 8)	106.9 ± 17.2 (n = 8)	265 ± 20.4 (n = 4)	150.2 ± 42.7 (n = 6)
ALP (U/L)	56.4 ± 9.6 (n = 9)d	85.1 ± 5.8 (n = 8)a	151.6 ± 14.3 (n = 8)b	94.0 ± 24.0 (n = 4)a	70.6 ± 12.6 (n = 6)
Total bilirubin (mg/dL)		0.5 ± 0.1 (n = 10)a	0.5 ± 0.1 (n = 19)a	0.8 ± 0.1 (n = 11)b
Sodium (mmol/L)	144.2 ± 0.5 (n = 4)	150.4 ± 1.8 (n = 7)	151.5 ± 1.1 (n = 10)
Potassium (mmol/L)	4.3 ± 0.1 (n = 4)	5.5 ± 0.9 (n = 7)	4.8 ± 0.8 (n = 10)
Magnesium (mg/dL)	2.0 ± 0.2 (n = 9)	2.4 ± 0.02 (n = 10)	2.5 ± 0.2 (n = 14)	2.9 ± 0.1 (n = 8)	2.2 ± 0.3 (n = 6)d
Calcium (mg/dL)	6.5 ± 0.6 (n = 9)	7.6 ± 0.4 (n = 10)a,b	6.4 ± 0.4 (n = 19)a	8.4 ± 0.2 (n = 9)b	5.9 ± 0.9 (n = 6)d
Chloride (mmol/L)	112 ± 0.7 (n = 4)	115.6 ± 1.9 (n = 7)
Creatine kinase (U/L)		1383.9 ± 236.8 (n = 8)	1002.3 ± 214.6 (n = 14)	2102.2 ± 722.8 (n = 5)

^a,b,cDifferent superscript letters indicate a significant (P < 0.05) difference between groups of male mice.

^dValues differ significantly (P < 0.05) between male and female mice within an experimental group.

As expected, compared with control mice, the DM mice had significantly (P < 0.05) higher blood glucose values, as well as significantly higher Hct and ALP activity. The glucose, creatinine, ALP, activity, and systolic, diastolic, and mean BP of DM mice were all significantly (P < 0.05) higher than those in DSS-treated mice. The heart rate and total bilirubin, total protein, albumin, and Hct values were significantly (P < 0.05) lower in DM mice than in DSS-treated mice.

Baseline sex-associated differences.

Differences between the male and female mice in the control and DSS-treated groups are presented in Table 2. In the control mice, body temperature, and ALP were all significantly (P < 0.05) greater in male than in female mice, while the heart rate was significantly (P< 0.05) lower in the male mice. As with the control mice, the DSS-treated mice male mice had elevated (P < 0.05) heart rates and body temperature values relative to those in the female mice. In this group, the male mice also had elevations (P < 0.05) at baseline in BUN, albumin, calcium, and ALT. As mentioned earlier, DM female mice were not tested in the current investigation due to the poor response of female mice to the streptozotocin treatment.^8,25,26

Effects of ABL.

Experiment 1.

The effects of the ABL on the control, DM, and DSS-treated mice are presented in Table 3 and in Figure 3. After ABL, the control mice demonstrated several significant (P < 0.05) changes at the 4-h time point, including decreases in body weight and activity. However, these changes were transient, because there were no significant differences between the baseline and 24-h values for these parameters. The body temperature remained normal at the 4-h time point in the female mice yet decreased at the 24-h time point in the male mice. The Hct was decreased significantly (P < 0.05) from the baseline value at both 4 and 24 h. The heart rate and lymphocyte count were decreased (P < 0.05) at the 4-h time point and subsequently were increased at the 24-h time point compared with the baseline value.

Table 3.

Effects of ABL on mice

	Control mice			DM mice			DSS mice
	Baseline	4 h	24 h	Baseline	4 h	24 h	Baseline	4 h	24 h
Body weight (g), P	27.8 ± 0.6 (n = 21)	27.1 ± 0.7a (n = 14)	27.4 ± 1.1 (n = 8)	22.8 ± 0.5 (n = 19)	21.4 ± 0.8a (n = 9)	21.7 ± 0.6a (n = 10)	21.6 ± 0.3 (n = 20)	21.6 ± 0.7 (n = 8)	19.3 ± 0.3a (n = 12)
Body temperature (°C), F	36.7 ± 0.2.9 (n = 11)	37.4 ± 0.4 (n = 11)					35.8 ± 0.4 (n = 6)	36.1 ± 0.4 (n = 6)
Body temperature (°C), M	38.3 ± 0.4 (n = 8)		37.6 ± 0.4a (n = 7)	37.6 ± 0.3 (n = 19)	36.5 ± 0.4 (n = 9)	35.8 ± 0.5a (n = 10)	37.8 ± 0.1 (n = 12)		35.5 ± 0.2a (n = 12)
Activity (no. of line breaks per 5 min), P	139 ± 11 (n = 17)	94 ± 10a (n = 14)	101 ± 9 (n = 5)	127 ± 8 (n = 19)	86 ± 10a (n = 9)	62 ± 7a (n = 10)	76 ± 5 (n = 14)	38 ± 3 (n = 6)	37 ± 7a (n = 7)
Heart rate (bpm), F	765 ± 11 (n = 11)	715 ± 16a (n = 11)					758 ± 9 (n = 6)	714 ± 16a (n = 6)
Heart rate (bpm), M	730 ± 5 (n = 8)		758 ± 5a (n = 8)	714 ± 11 (n = 18)	740 ± 7 (n = 9)	735 ± 9a (n = 10)	785 ± 7 (n = 12)		757 ± 6a (n = 11)
Systolic BP (mm Hg), P	161.9 ± 2.5 (n = 15)	163.2 ± 3.0 (n = 10)	150.8 ± 3.1 (n = 6)	161.2 ± 4.1 (n = 18)	140.6 ± 8.0a (n = 7)	135.3 ± 5.3a (n = 9)	148.4 ± 6.8 (n = 8)		143.5 ± 10.9 (n = 4)
Diastolic BP (mm Hg), P	130.2 ± 2.8 (n = 15)	125.7 ± 3.3 (n = 10)	122.7 ± 2.0 (n = 6)	128.0 ± 4.2 (n = 18)	94.8 ± 11.0a (n = 7)	99.7 ± 5.2a (n = 9)	106.8 ± 5.6 (n = 8)		104.4 ± 11.0 (n = 4)
Mean BP (mm Hg), P	140.5 ± 2.6 (n = 15)	137.9 ± 3.1 (n = 10)	131.8 ± 2.3 (n = 6)	139.9 ± 4.2 (n = 18)	109.9 ± 10.0a (n = 7)	111.2 ± 5.2a (n = 9)	120.3 ± 5.6 (n = 8)		117.1 ± 10.9 (n = 4)
Hct (%), P	42.8 ± 0.9 (n = 22)	34.1 ± 1.1a (n = 14)	32.3 ± 0.9a (n = 7)	46.6 ± 0.8 (n = 19)	35.0 ± 0.8a (n = 9)	35.0 ± 3.5a (n = 10)	57.8 ± 0.4 (n = 19)	49.8 ± 1.9a (n = 6)	43.6 ± 2.2a (n = 10)
WBC (× 103/µL), P	4.2 ± 0.6 (n = 22)	2.6 ± 0.3 (n = 14)	5.1 ± 1.7 (n = 7)	5.0 ± 0.8 (n = 19)	1.6 ± 2.0a (n = 9)	1.8 ± 0.5 (n = 10)	4.6 ± 0.8 (n = 19)	4.0 ± 1.5 (n = 6)	2.1 ± 0.8 (n = 10)
Neutrophils (× 103/µL), P	1.4 ± 0.4 (n = 19)	1.2 ± 0.2 (n = 14)	1.1 ± 0.6 (n = 5)	1.0 ± 0.2 (n = 19)	0.5 ± 0.1 (n = 9)	0.2 ± 0.1a (n = 10)	2.3 ± 0.5 (n = 15)	2.3 ± 1.2 (n = 5)	1.0 ± 0.5 (n = 10)
Lymphocytes (× 103/µL), P	2.9 ± 0.5 (n = 19)	1.3 ± 0.1a (n = 14)	5.0 ± 1.6a (n = 5)	4.0 ± 0.6 (n = 19)	1.0 ± 0.1a (n = 9)	1.5 ± 0.4 (n = 10)	1.7 ± 0.4 (n = 15)	0.7 ± 0.2 (n = 5)	1.1 ± 0.04 (n = 10)
Blood glucose (mg/dL), P	185 ± 15 (n = 21)	238 ± 14 (n = 14)	238 ± 11 (n = 7)	449 ± 24 (n = 16)	491 ± 29 (n = 8)	560 ± 25 (n = 10)	125 ± 9 (n = 19)	100 ± 16 (n = 8)	129 ± 11 (n = 11)
Total protein (mg/dL), P	5.0 ± 0.4 (n = 19)	4.6 ± 0.2 (n = 14)	4.8 ± 0.3 (n = 7)	4.8 ± 0.2 (n = 19)	3.9 ± 0.4 (n = 9)	4.8 ± 0.1 (n = 10)	6.2 ± 0.3 (n = 15)	6.8 ± 0.7a (n = 8)	6.6 ± 0.2 (n = 11)
Albumin (g/dL), P	2.0 ± 0.2 (n = 19)	1.6 ± 0.1a (n = 14)	1.9 ± 0.1 (n = 7)
Albumin (g/dL), F							1.9 ± 0.2 (n = 6)	1.8 ± 0.1 (n = 6)
Albumin (g/dL), M				1.8 ± 0.1 (n = 19)	1.5 ± 0.2 (n = 9)	2.2 ± 0.1 (n = 10)	2.5 ± 0.1 (n = 11)		2.8 ± 0.1 (n = 11)
BUN (mg/dL), P	26.5 ± 1.9 (n = 21)	30.1 ± 1.7 (n = 14)	30.7 ± 1.9 (n = 7)
BUN (mg/dL), F							39.5 ± 5.8 (n = 6)	79.8 ± 5.8a (n = 6)
BUN (mg/dL), M				23.0 ± 1.5 (n = 19)	27.6 ± 2.6 (n = 9)	31.0 ± 0.9 (n = 10)	55.2 ± 3.3 (n = 12)		61.6 ± 5.7a (n = 12)
Creatinine (mg/dL), P	0.26 ± 0.02 (n = 21)	0.31 ± 0.02 (n = 14)	0.18 ± 0.01 (n = 7)	0.29 ± 0.02 (n = 19)	0.27 ± 0.02 (n = 9)	0.22 ± 0.01 (n = 10)	0.23 ± 0.01 (n = 17)	0.38 ± 0.04a (n = 8)	0.24 ± 0.02 (n = 11)
ALT (U/L), P	99 ± 8 (n = 21)	132 ± 10 (n = 14)	94 ± 9 (n = 7)
ALT (U/L), F							83.8 ± 17.6 (n = 6)	148.7 ± 27.7 (n = 6)
ALT (U/L), M				114 ± 10 (n = 18)	118 ± 20 (n = 9)	79 ± 16a (n = 10)	103.4 ± 6.0 (n = 9)		135.1 ± 23.8 (n = 11)
ALP (U/L), P							80.0 ± 12.2 (n = 10)	65.0 ± 8.3 (n = 8)	133.7 ± 16.0 (n = 10)
ALP (U/L), F	56.4 ± 9.6 (n = 9)	79.7 ± 4.2 (n = 11)
ALP (U/L), M	86.8 ± 7.4 (n = 6)		80.8 ± 8.3 (n = 7)	151.6 ± 14.3 (n = 8)	140.5 ± 9.1 (n = 6)	218.7 ± 10.9a (n = 10)
AST (U/L), P	134.3 ± 23.4 (n = 21)	168.2 ± 21.4 (n = 13)	167.3 ± 37.5 (n = 7)	106.9 ± 17.2 (n = 15)	146.2 ± 22.3 (n = 9)	105.0 ± 6.2 (n = 10)	196.2 ± 31.9 (n = 20)	297.4 ± 104.6 (n = 8)	204.2 ± 27.2 (n = 8)
Sodium (mmol/L), P	146.3 ± 0.9 (n = 5)	147.5 ± 0.6 (n = 10)	148.6 ± 1.2 (n = 7)	151.5 ± 1.2 (n = 10)	150.7 ± 1.0 (n = 9)	150.0 ± 0.7 (n = 9)		176.0 ± 2.1 (n = 5)	174.6 ± 3.0 (n = 10)
Potassium (mmol/L), P	5.1 ± 0.6 (n = 11)	4.3 ± 0.1 (n = 12)	4.7 ± 0.5 (n = 7)	4.8 ± 0.8 (n = 10)	4.1 ± 0.3 (n = 9)	3.9 ± 0.1 (n = 9)		5.3 ± 1.3 (n = 5)	5.5 ± 0.4 (n = 10)
Magnesium (mg/dL), P	2.2 ± 0.1 (n = 19)	2.0 ± 0.1 (n = 14)	2.2 ± 0.1 (n = 8)	3.9 ± 1.0 (n = 15)	1.8 ± 0.1 (n = 9)	2.5 ± 0.1 (n = 10)	2.6 ± 0.2 (n = 14)	2.9 ± 0.3 (n = 8)	3.3 ± 0.1 (n = 11)
Chloride (mmol/L), P	114.3 ± 1.3 (n = 11)	118.2 ± 1.0a (n = 12)	116 ± 1.4 (n = 7)	115.2 ± 2.0 (n = 10)	119.0 ± 1.1 (n = 9)	115.7 ± 0.6 (n = 10)
Calcium (mg/dL), F							5.9 ± 0.9 (n = 6)	5.2 ± 0.9 (n = 6)
Calcium (mg/dL), M				6.4 ± 0.4 (n = 19)	6.4 ± 0.6 (n = 9)	8.9 ± 0.2a (n = 10)	8.4 ± 0.2 (n = 9)		9.2 ± 0.2a (n = 12)

Data presented are pooled (P) from both sexes or are separated according to sex (M, male; F, female) when significant sex-associated effects were detected at baseline. All data for diabetic mice are from male mice only.

^aSignificant difference from the baseline value within the experimental group.

Figure 3.

Changes in (A) body weight, (B) body temperature, (C) activity, (D) heart rate, and (E) Hct in the 3 experimental groups over time. Body weight, activity, and hematocrit data include male and female mice both; body temperature and heart rate data were obtained from male mice only. #, Value significantly (P < 0.05) different from the baseline for the control group; *, Value significantly (P < 0.05) different from the baseline value within the experimental group.

Both experimentally compromised groups of mice demonstrated more adverse effects associated with ABL than did the control mice. The DSS-treated mice had significant (P < 0.05) decreases in body weight, body temperature, Hct, and activity. These mice also showed signs of increased dehydration, because their azotemia worsened at both the 4- and 24-h time points. The total protein concentration was elevated (P < 0.05) at the 4-h time point but returned to near baseline concentrations by 24 h after the ABL. Interestingly, the heart rate of this group of mice declined significantly (P < 0.05) at both the 4- and 24-h time points when compared with the baseline rate, despite the apparent progression of the hypovolemia. The WBC count was significantly (P < 0.05) decreased at the 24-h time point. The plasma calcium concentration was significantly (P < 0.05) elevated 24 h after the blood loss. The sodium concentrations at the 4-and 24-h time points in the DSS-treated mice were significantly (P < 0.05) higher than those in the other 2 experimental groups. Baseline analysis of the sodium concentration in the DSS-treated mice frequently was not possible due to the small plasma volume collected from the hemoconcentrated blood of this group at the baseline time point.

Similar to the DSS-treated mice, the DM mice had significant (P < 0.05) drops in body weight and activity at both the 4- and 24-h time points and a decrease in the body temperature at the 24-h time point. The systolic, diastolic, and mean BP values in these mice decreased (P < 0.05) significantly at both 4 and 24 h from baseline, whereas the heart rate increased (P < 0.05) at the 24-h time point. On CBC analysis, the Hct decrease in DM mice was similar to the change in control mice; DM mice also showed a transient drop in the WBC and lymphocyte counts at the 4-h time point and in the neutrophil count at the 24-h time point. At the 24 h time point, the ALP concentration was elevated (P < 0.05) compared with the baseline, whereas the ALT activity was decreased (P < 0.05).

Experiment 2.

The results for Experiment 2 are presented in Tables 4, 5, and 6 and in Figure 4. Fluids were administered at 2 different time points—either immediately after or 30 min before the mice were anesthetized for blood collection—thus allowing for determination of the effects of intraperitoneal lactated Ringers solution on CBC and chemistry values independent of ABL. The other dependent variables (body weight and temperature, activity, heart rate, and BP parameters) were measured before fluid administration. In the control mice, administration of lactated Ringers solution before ABL significantly (P < 0.05) decreased BUN and increased the plasma ALP activity and magnesium concentrations. The administration of fluids significantly (P < 0.05) decreased the plasma BUN and potassium concentrations in the DM mice, bringing their plasma concentrations of potassium to similar levels as in control mice. Similarly, fluid administration to dehydrated DSS-treated mice significantly (P < 0.05) decreased BUN, total protein, albumin, and sodium levels, returning BUN concentrations to levels found in healthy control mice before fluid administration.

Table 4.

Effects of ABL and fluid replacement in control mice

	Fluids provided immediately after ABL		Fluids provided 30 min before ABL
	Baseline	24 h	Baseline	24 h
Body weight (g)	26.6 ± 0.8 (n = 8)	25.9 ± 0.6 (n = 7)a	24.5 ± 1.7 (n = 7)	23.6 ± 1.6 (n = 8)a
Body temperature (°C)	37.6 ± 0.4 (n = 8)	37.8 ± 0.3 (n = 8)	37.8 ± 0.5 (n = 4)	37.5 ± 0.4 (n = 4)
Activity (no. of line breaks per 5 min)	166 ± 12 (n = 7)	146 ± 15 (n = 8)	148 ± 11 (n = 7)	114 ± 14 (n = 7)
Heart rate (bpm)	751 ± 10 (n = 8)	758 ± 8 (n = 8)	746 ± 18 (n = 7)	768 ± 14 (n = 7)
Systolic BP (mm Hg)	143 ± 12 (n = 8)	124 ± 12 (n = 8)a	109 ± 13 (n = 6)	111 ± 10 (n = 5)
Diastolic BP (mm Hg)	116.9 ± 11.8 (n = 8)	94.3 ± 11.8 (n = 8)	82.2 ± 11.8 (n = 6)	86.1 ± 9.8 (n = 5)
Mean BP (mm Hg)	125.4 ± 11.9 (n = 8)	103.8 ± 11.6 (n = 8)	90.8 ± 12.2 (n = 6)	94.1 ± 9.9 (n = 5)
Hct (%)	42.2 ± 1.6 (n = 8)	30.0 ± 1.3 (n = 8)a	43.0 ± 1.1 (n = 7)	29.5 ± 0.7 (n = 7)a
WBC (× 103/µL)	3.4 ± 0.9 (n = 8)	4.4 ± 1.0 (n = 8)	3.0 ± 0.9 (n = 7)	2.3 ± 0.2 (n = 7)
Neutrophils (× 103/µL)	1.1 ± 0.5 (n = 8)	1.0 ± 0.4 (n = 7)	1.7 ± 0.8 (n = 7)	0.5 ± 0.5 (n = 7)
Lymphocytes (× 103/µL)	2.3 ± 0.4 (n = 8)	3.3 ± 0.8 (n = 7)	1.3 ± 0.1 (n = 7)	1.8 ± 0.2 (n = 7)
Blood glucose (mg/dL)	182 ± 11 (n = 8)	199 ± 18 (n = 8)	197 ± 18 (n = 7)	270 ± 39 (n = 7)
BUN (mg/dL)	27.1 ± 1.1 (n = 8)	27.9 ± 2.1 (n = 8)	18.9 ± 2.6 (n = 7)b	30.0 ± 1.5 (n = 7)a
Creatinine (mg/dL)	0.36 ± 0.05 (n = 8)	0.25 ± 0.03 (n = 8)	0.27 ± 0.02 (n = 7)	0.27 ± 0.02 (n = 7)
ALT (U/L)	146 ± 19 (n = 8)	152 ± 24 (n = 8)	131 ± 14 (n = 7)	188 ± 31 (n = 6)
AST (U/L)	103.0 ± 17.0 (n = 7)	93.1 ± 12.7 (n = 8)	138.8 ± 22.0 (n = 5)	128.1 ± 22.1 (n = 8)
ALP (U/L)	82.6 ± 7.3 (n = 7)	65.5 ± 4.8 (n = 8)	121.2 ± 7.0 (n = 6)b	127.0 ± 43.4 (n = 7)
Sodium (mmol/L)	147.8 ± 1.0 (n = 5)	148.5 ± 0.7 (n = 8)	147.3 ± 0.9 (n = 8)	150.3 ± 0.5 (n = 7)
Potassium (mmol/L)	4.4 ± 0.2 (n = 5)	4.4 ± 0.1 (n = 8)	4.4 ± 0.1 (n = 7)	4.5 ± 0.1(n = 7)
Magnesium (mg/dL)	2.7 ± 0.2 (n = 8)	2.4 ± 0.2 (n = 8)	3.2 ± 0.2 (n = 6)b	2.5 ± 0.1 (n = 7)a
Calcium (mg/dL)	8.0 ± 0.4 (n = 8)	8.1 ± 0.3 (n = 8)	8.6 ± 0.6 (n = 6)	7.3 ± 0.6 (n = 7)

^aValue significantly (P < 0.05) different from that at baseline within the experimental group.

^bValue significantly (P < 0.05) different from that at baseline when fluids were given immediately after ABL.

Figure 4.

BUN concentrations in the DSS-treated mice in experiments 1 and 2. * denotes a significant difference between the baseline value and the time point within that group. # denotes a significant difference between the baseline values for the mice receiving fluids before and immediately after the ABL.

Table 5.

Effects of ABL and fluid replacement in DM mice

	Fluids provided immediately after ABL		Fluids provided 30 min before ABL
	Baseline	24 h	Baseline	24 h
Body weight (g)	20.1 ± 0.8 (n = 12)	18.8 ± 0.9 (n = 12)a	21.8 ± 0.7 (n = 9)	20.3 ± 0.6 (n = 9)a
Body temperature (°C)	35.5 ± 0.5 (n = 10)	34.8 ± 0.5 (n = 11)	37.1 ± 0.7 (n = 7)	37.2 ± 0.4 (n = 7)
Activity (no. of line breaks per 5 min)	114 ± 14 (n = 12)	73 ± 13 (n = 12)a	105 ± 9 (n = 9)	76 ± 12 (n = 9)a
Heart rate (bpm)	697 ± 18 (n = 12)	700 ± 12 (n = 12)	697 ± 8 (n = 9)	672 ± 12 (n = 9)
Systolic BP (mm Hg)	109 ± 5 (n = 10)	95 ± 4 (n = 11)a	136 ± 9 (n = 9)	130 ± 7 (n = 9)
Diastolic BP (mm Hg)	81.6 ± 4.3 (n = 10)	70.6 ± 3.8 (n = 11)a	107.0 ± 8.8 (n = 9)	100.0 ± 6.6 (n = 9)
Mean BP (mm Hg)	90.5 ± 4.4 (n = 10)	78.6 ± 3.8 (n = 11)a	116.4 ± 8.9 (n = 9)	109.7 ± 6.6 (n = 9)
HCT (%)	43.0 ± 2.0 (n = 12)	35.1 ± 1.2 (n = 12)a	47.20 ± 1.1 (n = 9)	32.6 ± 2.1 (n = 9)a
WBC (× 103/µL)	4.8 ± 0.7 (n = 12)	3.1 ± 1.2 (n = 12)	3.6 ± 0.6 (n = 9)	2.9 ± 0.6 (n = 9)
Neutrophils (× 103/µL)	1.1 ± 0.3 (n = 12)	1.5 ± 0.6 (n = 7)	1.1 ± 0.2 (n = 8)	0.5 ± 0.1 (n = 7)a
Lymphocytes (× 103/µL)	3.7 ± 0.5 (n = 8)	1.6 ± 0.6 (n = 7)a	2.5 ± 0.5 (n = 8)	2.3 ± 0.5 (n = 9)
Blood glucose (mg/dL)	434 ± 33 (n = 12)	483 ± 33 (n = 11)	400 ± 13 (n = 9)	446 ± 25 (n = 8)
Total protein (mg/dL)	5.8 ± 0.7 (n = 6)	4.4 ± 0.2 (n = 6)
Albumin (g/dL)	2.0 ± 0.3 (n = 6)	2.1 ± 0.1 (n = 6)
BUN (mg/dL)	33.6 ± 3.5 (n = 12)	32.5 ± 1.5 (n = 11)	18.8 ± 1.0 (n = 9)b	29.1 ± 1.2 (n = 8)a
Creatinine (mg/dL)	0.4 ± 0.05 (n = 5)	0.2 ± 0.01 (n = 12)a	0.3 ± 0.1 (n = 5)	0.3 ± 0.1 (n = 9)
ALT (U/L)	207 ± 72 (n = 12)	210 ± 87 (n = 5)	179 ± 26 (n = 9)	135 ± 16 (n = 6)a
AST (U/L)	139.6 ± 41.0 (n = 5)	196.3 ± 28.0 (n = 12)	129.0 ± 23.2 (n = 4)	142.1 ± 24.1 (n = 9)
ALP (U/L)	161.2 ± 22.5 (n = 5)	182.1 ± 14.5 (n = 12)	168.5 ± 25.0 (n = 4)	161.2 ± 22.5 (n = 9)
Sodium (mmol/L)	148.4 ± 1.4 (n = 9)	149.0 ± 1.4 (n = 11)	151.2 ± 0.6 (n = 7)	152.2 ± 1.2 (n = 9)
Potassium (mmol/L)	6.5 ± 0.8 (n = 9)	4.3 ± 0.2 (n = 11)a	4.3 ± 0.1 (n = 7)b	4.6 ± 0.1 (n = 9)a
Magnesium (mg/dL)	3.1 ± 0.5 (n = 5)	2.5 ± 0.1 (n = 12)	3.3 ± 0.1 (n = 3)	2.8 ± 0.1 (n = 9)
Calcium (mg/dL)	8.8 ± 1.0 (n = 11)	9.0 ± 0.1 (n = 12)	8.0 ± 0.4 (n = 7)	7.8 ± 0.4 (n = 9)

^aValue significantly (P < 0.05) different from that at baseline within the experimental group.

^bValue significantly (P < 0.05) different from that at baseline when fluids were given immediately after ABL.

Table 6.

Effects of ABL and fluid replacement in DSS-treated mice

	Fluids provided immediately after ABL			Fluids provided 30 min before ABL
	Baseline	4 h	24 h	Baseline	24 h
Body weight (g)	21.2 ± 0.2 (n = 22)	21.8 ± 0.3a (n = 12)	20.6 ± 0.4a (n = 10)	21.6 ± 0.6 (n = 10)	21.1 ± 0.6a (n = 10)
Body temperature (°C)	36.8 ± 0.2 (n = 19)	35.5 ± 0.3 (n = 10)	36.6 ± 0.4 (n = 8)	37.2 ± 0.4 (n = 10)	36.6 ± 0.3 (n = 10)
Activity (no. of line breaks per 5 min)	129 ± 12 (n = 15)	69 ± 13a (n = 6)	48 ± 6a (n = 10)	143 ± 14 (n = 10)	58 ± 4a (n = 10)
Heart rate (bpm)	775 ± 3 (n = 21)	733 ± 7 a (n = 10)	781 ± 4 (n = 12)	772 ± 6 (n = 10)	771 ± 7 (n = 10)
Systolic BP (mm Hg)	136 ± 4 (n = 18)		133 ± 7 (n = 10)	141 ± 10 (n = 10)	133 ± 8 (n = 9)
Diastolic BP (mm Hg)	99.3 ± 5.6 (n = 19)		103.4 ± 7.2 (n = 10)	107.5 ± 9.4 (n = 10)	102.2 ± 7.4 (n = 10)
Mean BP (mm Hg)	110.6 ± 5.7 (n = 19)		113.1 ± 7.0 (n = 10)	118.5 ± 9.6 (n = 10)	112.1 ± 7.8 (n = 10)
Hct (%)	54.3 ± 0.5 (n = 13)	37.9 ± 3.4a (n = 4)	40.3 ± 0.6a (n = 9)	52.0 ± 1.1 (n = 10)	38.9 ± 1.6a (n = 10)
WBC (× 103/µL)	3.1 ± 0.7 (n = 13)	4.1 ± 0.4 (n = 4)	1.9 ± 0.5 (n = 9)	1.3 ± 0.2 (n = 10)	0.9 ± 0.1 (n = 10)
Neutrophils (× 103/µL)	0.8 ± 0.2 (n = 13)	2.0 ± 0.5 (n = 3)	0.3 ± 0.1 (n = 9)	0.5 ± 0.1 (n = 10)	0.2 ± 0.1a (n = 9)
Lymphocytes (× 103/µL)	2.3 ± 0.6 (n = 13)	1.8 ± 0.4 (n = 3)	1.5 ± 0.4 (n = 9)	0.8 ± 0.2b (n = 10)	0.7 ± 0.1 (n = 9)
Blood glucose (mg/dL)	133 ± 9 (n = 21)	120 ± 7 (n = 12)	161 ± 5 (n = 10)	136 ± 21 (n = 10)	172 ± 10 (n = 10)
Total protein (mg/dL)	7.5 ± 0.4 (n = 16)	6.1 ± 0.4a (n = 10)	6.4 ± 0.4 (n = 6)	6.1 ± 0.2b (n = 10)	5.7 ± 0.1 (n = 10)
Albumin (g/dL)	2.6 ± 0.1 (n = 16)	1.8 ± 0.1a (n = 6)	2.2 ± 0.1a (n = 10)	2.1 ± 0.1b (n = 10)	2.1 ± 0.1 (n = 10)
BUN (mg/dL)	50.0 ± 3.6 (n = 21)	40.2 ± 1.8a (n = 12)	41.1 ± 2.8 (n = 10)	37.9 ± 1.8b (n = 10)	37.8 ± 1.1 (n = 10)
ALT (U/L)	62 ± 5 (n = 8)	173 ± 19 (n = 9)	87 ± 11 (n = 10)	90 ± 16 (n = 10)	143 ± 37 (n = 10)
AST (U/L)	127.0 ± 16.9 (n = 4)	276.3 ± 50.6 (n = 9)	98.8 ± 15.7 (n = 5)	136.0 ± 28.2 (n = 6)	212.1 ± 30.5 (n = 9)
ALP (U/L)	88.0 ± 8.3 (n = 4)	107.4 ± 12.8 (n = 9)	62.2 ± 5.2 (n = 5)	102.0 ± 10.1 (n = 7)	94.7 ± 15.8 (n = 9)
Sodium (mmol/L)	169.8 ± 3.7 (n = 7)	171.5 ± 1.7 (n = 11)	161.8 ± 1.0 (n = 10)	160.5 ± 1.0b (n = 9)	162.9 ± 1.5 (n = 10)
Potassium (mmol/L)	5.6 ± 0.4 (n = 3)	4.7 ± 0.2 (n = 11)	4.5 ± 0.2 (n = 10)	4.8 ± 0.2 (n = 6)	4.5 ± 0.1 (n = 10)
Magnesium (mg/dL)	2.4 ± 0.1 (n = 8)		1.7 ± 0.1a (n = 10)	2.4 ± 0.10 (n = 9)	2.3 ± 0.1 (n = 10)
Calcium (mg/dL)	8.3 ± 0.2 (n = 15)	7.7 ± 0.2 (n = 11)	6.6 ± 0.3 (n = 10)	7.9 ± 0.5 (n = 10)	8.6 ± 0.3 (n = 10)

^aValue significantly (P < 0.05) different from that at baseline within the experimental group.

^bValue significantly (P < 0.05) different from that at baseline when fluids were given immediately after ABL.

Effects of fluid replacement on ABL.

The body weights and Hct of the control mice were significantly (P < 0.05) lower in mice receiving either type of fluid treatment when assessed 24 h after ABL. The BUN was significantly (P < 0.05) higher at the 24-h time point in the mice that received fluids prior to blood collection, achieving values similar to the control mice that had not received fluids before the ABL. The magnesium concentration was significantly (P < 0.05) lower in mice given fluids before ABL, again to concentrations similar to those in the control mice at baseline. Lastly, the systolic BP was significantly (P < 0.05) lower in the mice treated with fluids after ABL. In contrast, mice that received fluids before ABL demonstrated no significant change in systolic BP at the 24-h time point. The mean and diastolic BP values did not change at the 24-h time point in association with either type of fluid administration.

The changes in the Hct, body weight, and activity of DSS-treated mice after ABL and fluid therapy were similar to those of the untreated mice in experiment 1. However, administration of lactated Ringers solution after ABL decreased (P < 0.05) the BUN at the 4-h time point, and fluid administration before ABL maintained the decreased BUN through the 24-h time point. The administration of lactated Ringers solution to the mice after ABL decreased the heart rate at the 4-h time point (P < 0.05). The administration of fluids at both time points brought the total protein and albumin concentrations to near-normal levels in the dehydrated mice at the 24-h time point. There was a significant (P < 0.05) decrease in the magnesium concentration at the 24-h time point in the mice given fluids after ABL. Lastly, the administration of fluids before the ABL resulted in a decrease in the neutrophil count at the 24-h time point (P < 0.05).

The administration of fluids at either time point did not prevent a significant loss of body weight and activity at 24 h after the baseline ABL in the DM mice (P < 0.05). The changes at the 24-h time point in the Hct and BUN values after the provision of intraperitoneal lactated Ringers solution were similar to the control mice. The ABL dramatically decreased the Hct at the 24-h time point, independent of when the mouse received the fluids. The BUN increased significantly (P < 0.05) over the 24 h in the group given fluids before ABL, but the values at the 24-h time point were similar to those of the mice that received fluids after ABL and were not indicative of azotemia. In addition, the mice treated with fluids after ABL had a significant (P < 0.05) decrease in the creatinine concentration at the 24-h time point compared with the baseline. All 3 BP measurements were significantly (P < 0.05) decreased compared with the baseline values in the mice given fluids after ABL, whereas mice that received fluids before the ABL had no change in BP.

Discussion

In the current study, we imposed a significant physiologic challenge modeling an acute blood loss of approximately 15% of the total blood volume in mice with the common underlying clinical conditions of DM and dehydration. Baseline testing demonstrated that both DM and DSS-treated mice were compromised before ABL, and ABL affected them to a greater extent than it did the healthy control mice. Despite these effects, there were no ABL-induced deaths in any of our mice. We then attempted to ameliorate the observed negative effects of ABL by using 1.5 mL lactated Ringers solution, a crystalloid physiologic fluid replacement therapy. By administering lactated Ringers solution intraperitoneally either 30 min before or immediately after the ABL, we were able to ameliorate some, but not all, of the adverse changes induced by the ABL. These results demonstrate that although the loss of a significant volume of blood for diagnostic bloodwork does negatively affect mice, it was not fatal within 24 h of ABL, and some of the negative effects could be overcome by the administration of intraperitoneal fluid therapy.

Several of the plasma chemistry values of the control mice were not within the normal ranges reported for the blood analysis system we used in the current study or in the reference ranges provided by other published studies, demonstrating the importance of determining baseline values under each experimental condition.^11,41,44,50 Published values for clinically healthy mice vary widely in the literature. For example, in our study, the mean values for the WBC count was 3.3 × 10³ cells/dL in normal male mice and 5.0 × 10³ cells/dL for normal female mice. These values are consistent with the values (6.6 and 4.60 × 10³ cells/dL, respectively) in one study⁴¹ but are profoundly different from those (12.530 × 10³ cells/μL and 12.80 × 10³ cells/μL) reported elsewhere.⁴⁴ The numbers of neutrophils did not dramatically differ among the 3 studies;^41,44 and the differences in the numbers of lymphocytes accounted for the reported differences in WBC. In addition, the mean calcium values reported by one of the cited studies⁴⁴ do not fall in the normal range reported by the reference laboratory we used in the current investigation. Ultimately, there are many factors that can affect the blood values of mice, including strain, sex, circadian variability, type of anesthesia used for sedation, and sampling site, and all of these factors need to be considered when using published reference ranges for mice. In addition, the BP measures in our studies were higher than is typically reported in the literature for C57BL/6 mice.²⁰ This increase in likely due to the nature of the testing method used. Despite the multiple acclimation sessions before the actual testing, mice are easily stressed by handling, potentially accounting for the elevated BP results in the current study.

Our cumulative data suggest that both DM and DSS-treated mice were physiologically compromised prior to ABL, and DSS-treated mice appear compromised more severely than are the DM mice. Both groups weighed less than did the control mice, and the DSS-treated mice also showed decreased activity. Interestingly, the nature of the muscle wasting and poor body condition appeared to be different between these 2 groups. Based on the history and physical exam of the mice, the weight loss in the DM mice was due primarily to muscle and fat wasting associated with the streptozotocin treatment and the subsequent reduced rate of weight gain over several weeks. In contrast, the weight loss in the DSS-treated mice was acute and due to a combination of both dehydration and muscle wasting.

Diabetes in DM mice was induced approximately 4 wk prior to our current studies. As expected, destruction of the islet cells resulted in significant elevations of baseline measures of blood glucose and ALP activity. In addition, the weight loss or failure to gain weight exhibited by the DM mice is not surprising, given that insulin is the most potent anabolic hormone, promoting both increased protein synthesis and inhibiting protein degradation.¹² Although several studies have tested the physiologic effects of streptozotocin treatment in C57BL/6 mice, its effects on cardiovascular parameters in this strain are not well defined. In contrast to a previous study⁵² that reported a significant increase in systolic BP, streptozotocin treatment in the current investigation and several other studies did not significantly affect baseline BP in C57BL/6 mice.^13,17,22 Interestingly, diabetes in humans is associated with hypertension.^1,18,49 Whereas streptozotocin treatment has been shown to consistently decrease heart rate in rats, 3 of the 5 studies reporting the effect of streptozotocin on the heart rate of C57BL/6 mice report a drop in heart rate,^13,17,27 and the current study and a previous report⁵² showed no significant change. The response to streptozotocin treatment on the immune system is strain-specific in mice and does not affect immune responses in C57BL/6 mice.^5,36 Consistent with these earlier studies, we saw no significant differences between the control and DM mice in baseline WBC counts or differentials. Although our DM mice had significant weight reduction, they did not appear to have developed additional complications in the 4 wk after the induction of diabetes.

The initial weight loss observed in the DSS-treated mice was due to both muscle wasting and dehydration. Muscle wasting and loss of body condition can happen rapidly in mice in a negative energy balance, due to their high metabolism.^39,45 Differentiating acute weight loss due to a significant negative energy from a chronic failure to gain muscle mass, as occurred in the DM mice, could be very valuable in identifying the underlying cause of the pathology. Unfortunately, differentiating between these 2 types of muscle wasting was difficult on physical exam. Although the serum chemistry of DSS-treated mice suggested marked dehydration at baseline, signs of dehydration were not overt upon physical exam. The physical exam diagnosis of dehydration is very difficult in many species, including mice, because the signs (for example weakness, piloerection, and sunken eyes) are often subjective and nonspecific until the dehydration becomes severe. The more specific sign of dehydration, skin tenting, occurs when dehydration is moderate to severe.⁵ DSS-induced dehydration resulted in several classic changes in cardiovascular and plasma chemistry parameters, including an elevated heart rate and decreased BP, and elevations in Hct, total protein, albumin, and BUN. The cardiovascular changes, combined with evidence of azotemia (likely prerenal in nature, considering the experimental design of the study, physical exam, bloodwork findings, and positive response to fluid administration) in these mice is an important finding, given that these pathologies are associated with lethargy and inappetence in other species, potentially accounting for the weight loss and decreased activity seen in this group at baseline. The rapid onset of clinical and hematologic signs in the C57BL/6J mice with DSS-treatment is consistent with previous research, which demonstrated enhanced sensitivity of C57BL/6 mice to DSS relative to many other strains of mice.^28,31 Ultimately, the baseline comparisons with the control mice demonstrated that both DM and DSS-treated mice were physiologically compromised.

As expected, the ABL had transient effects on the healthy control mice, with more severe signs of morbidity in DM and DSS-treated mice. The control mice had small changes in body weight, activity, heart rate, lymphocyte count, and albumin at the 4-h time point, all of which rebounded to baseline or above by 24 h after ABL. All 3 groups of mice had a significant drop in the Hct after ABL.³⁵ This drop is consistent with other studies of ABL in mice but is in stark contrast to the response to ABL in dogs, in which splenic contraction after ABL maintains RBC levels despite moderate blood loss. There are significant differences between the spleen in the dog and mouse. Compared with mice, dogs have a sinusoidal spleen, which can store greater volumes of erythrocytes and has more splenic smooth muscle, thus enabling the organ to contract more rapidly and deliver greater volumes of erythrocytes.⁶ One surprising finding in all 3 experimental groups of mice was that there was no decrease in the total protein after ABL, and the dehydrated DSS-treated mice actually showed a significant increase in the total protein at the 4-h time point, potentially due to an increase in plasma acute-phase proteins released in response to ABL.²³ Because significant blood loss typically results in a decrease in total plasma protein, a transient drop in total protein may have occurred early after the ABL in all 3 animal models, with subsequent compensation and recovery by the 4-h time point.

Both DM and DSS-treated mice had significant decreases in body weight, temperature, and activity at 24 h after ABL; these changes were not present in the control mice. All 3 of these signs are nonspecific indicators of impaired clinical health and, interestingly, appear to have different mechanisms in the diseased groups. The DSS-treated mice showed several changes that cumulatively can explain their worsening clinical condition. The azotemia that was present in these dehydrated mice at baseline progressed after ABL, with BUN values exceeding 70 mg/dL at the 4-h time point and remaining elevated over baseline at the 24-h time point. In addition, the DSS-treated mice had a significant drop in heart rate over the 24 h after ABL. This decrease is likely due to the biphasic sympathoinhibitory response seen in severe hemorrhage.^4,21,43 In healthy animals with hemorrhage of less than approximately 30% blood loss, there is an increase in sympathetic tone that increases heart rate and maintains blood pressure to maintain cardiac output. At greater percentages of blood loss, there is a protective inhibition of sympathetic tone, resulting in a rapid drop in heart rate and blood pressure. Although the blood loss in the current study was well below 30% of the total blood volume, the mice were significantly compromised and dehydrated before ABL, potentially leading to the inhibition of the sympathetic response at a lower percentage of blood loss. Combined with the observed drop in Hct, both azotemia and decreased heart rate may have contributed to the lethargy and weight loss seen in the DSS-treated mice. Because neurohormonal mechanisms activated by hypovolemia, including arginine vasopressin and cortisol release, activation of the renin–angiotensin–aldosterone system, and increased sympathetic tone,^12,16 may have been activated by the preceding dehydration in these mice, their ability to maintain homeostasis in the face of the additional physiologic challenge appears limited. Given that the condition of these mice deteriorated between the 4-h and 24-h time points, it remains possible that deterioration could have progressed beyond the 24-h time point, potentially resulting in the death of the mouse or the necessity of euthanasia.

In the DM mice, the observed pathologic changes in animals appear to be driven by changes in BP and Hct. As in the DSS-treated mice, the DM mice had a significant drop in Hct after ABL. DSS-treated mice also had a significant decrease in BP at the 4- and 24-h time points and an increase in the heart rate at 24 h. The drop in blood pressure is unlikely to be due to hypovolemia or dehydration, because the plasma chemistry analysis did not reveal changes consistent with dehydration. As we alluded to earlier, diabetes and elevated glucose reduce BP variability in humans, mice, and rats, with human and rat studies also showing changes in heart rate variability, both of which are indicative of an autonomic neuropathy.^{4,18,24,27,47} Previous work in streptozotocin-treated C57BL/6 mice has demonstrated evidence of cardiovascular autonomic neuropathy or dysfunction,^27,52 which is an important and significant DM complication in humans.²⁴ Another study⁴ found that a drop in blood pressure occurred at lower volumes of blood loss in DM rats than in control rats, due to a blunted sympathetic response and an augmented parasympathetic response to blood loss. Such diabetic cardiovascular autonomic neuropathy may limit a diabetic animal's ability to respond to ABL, leading to signs of systemic disease more rapidly than in a clinically healthy animal.

Although the negative effects of ABL were not fatal in any of the mice in the current study, one mouse treated with DSS progressed to moribundity that would have required humane euthanasia if the animal were not already at its experimental endpoint. The lethargy or decreased activity described in other mice did not progress to the humane endpoint criteria, according to the IACUC protocol expectations. Our study verifies the importance of individual monitoring of animals, because any procedure can be associated with a range of morbidity, potentially resulting in criteria that necessitate invoking the humane endpoint for the animal in the study. In addition, the effect of the blood loss required for diagnostic bloodwork on the experimental use of the animals needs to be considered, because the associated ABL may render future data from the animal invalid for the goals of the project. Although study subjects ideally should contribute maximally to the research mission, investigators and clinicians must temper scientific objectives against the individual welfare of each animal. Our work cautions that ABL may result in certain mice reaching predetermined, IACUC-approved humane endpoints prior to their planned experimental endpoints.

The design of the second set of experiments allowed us to test the effects of fluid therapy on the results of CBC and serum chemistry analyses in mice with either DM or DSS-induced dehydration. Not surprisingly, the most profound changes occurred in the dehydrated, DSS-treated mice, which showed clinical improvements in several markers of dehydration in just 30 min after fluid administration, including decreases in BUN, total protein, albumin, and sodium. Interestingly, the hemodilution of the plasma proteins was not associated with a proportional drop in Hct. In most species, fluids are administered intravenously to increase the intravascular fluid volume rapidly. In these dehydrated mice, however, we observed rapid absorption of fluids, leading to clinical improvement in the biomarkers associated with dehydration.

The administration of intraperitoneal fluids decreased BUN concentrations to below baseline values in both the control and DM mice, demonstrating a significant diuresis effect in the normally hydrated animals. The BUN values returned to normal baseline by the 24-h time point, despite ABL. Diuresis was evident in the DM mice also, which showed a significant decrease in potassium levels. Although most diabetic animals have a loss of total body potassium,⁹ the movement of fluids and potassium from the intracellular space to the extracellular space can result in hyperkalemia. Fluid administration then dilutes the potassium concentration, giving a more accurate assessment of true total-body potassium levels.⁹ Intraperitoneal fluid therapy has been used successfully in human medicine to treat dehydration when intravenous access is limited, because the peritoneal lining is permeable to both fluids and small molecules.^2,14,30,51

The administration of lactated Ringers solution, either before or after ABL, ameliorated some of the negative effects caused by ABL but not all of them. Although the changes in the control mice after ABL were mild, the administration of fluids at the time of the ABL did prevent the loss of body temperature and changes in heart rate and lymphocyte count seen at 24 h The time of fluid administration did not appear to alter these responses. One surprising result after fluid administration was a statistically significant drop in body weight in the control mice at the 24-h time point, when there was no significant change when fluids were not administered. The underlying mechanism for this change is not readily apparent. Not surprisingly, the significant drop in the Hct value was not affected by the fluid administration, given that the fluids further dilute the remaining circulating RBC.

In the DM mice, the administration of fluids prevented some adverse physiologic effects but was inadequate to completely eliminate negative effects associated with ABL. Although the fluids prevented the significant drop in body temperature at the 24-h time point that occurred in the absence of fluid therapy, body weight and activity still decreased. The failure of fluids to maintain the weight of the DM mice may be related to the polyuria and polydipsia associated with DM, which would prevent the fluid retention required to maintain their body weight. Similarly, these effects may permit the significant drop in BP when fluids were administered after ABL. Interestingly, the administration of fluids 30 min before ABL in the DM mice appeared to have a protective effect, preventing the drop in BP. In agreement with previous studies, our current findings indicate that improving the animals’ condition with pre-emptive fluids at the time of blood loss helps mitigate the negative effects of the procedure.^15,46,53

The administration of fluids to the dehydrated DSS-treated mice dramatically improved the dehydration-induced azotemia and abrogated the negative effects of blood loss on the heart rate, significantly improving both of these parameters after ABL. In addition, although these positive changes partially prevented the drop in body temperature at the 24-h time point, they did not appreciably diminish the decrease in body weight and activity, similar to the changes seen in DM mice. Most of the fluids had likely been absorbed into the blood stream by the 4-h time point, with the consequent diuresis resulting in the weight gain of the mice at this time point. Although there is a predicted net gain of approximately 1.25 mL of liquids at the time of blood loss (250 μL of blood lost and 1.5 mL of fluids administered), mice gained only 0.6 g at the 4-h time point.

In conclusion, the current investigation was designed to assess the effect of ABL for diagnostic bloodwork on 2 models of clinically ill mice. These pathologies lead to significant changes in the general health of the mice and alter parameters of the diagnostic bloodwork and cardiovascular system. Although the healthy, control mice had mild transient changes in response to an ABL like that for diagnostic bloodwork, the DM and DSS-treated mice had prolonged decreases in body weight and in temperature, activity, and cardiovascular parameters as well as a progression of azotemia in the dehydrated mice. Although none of these effects were fatal in the mice in the current study, the negative changes show that ABL affected the physical condition and overall welfare of the mice; therefore, physiologic changes need to be considered for determination of both the experimental and humane clinical endpoints. Our results demonstrate that the provision of intraperitoneal fluid therapy can ameliorate many of the pathologic changes associated with ABL and should be considered as a preventative health measure for laboratory mice.