Cerebrovascular accident is one of the major causes of disability among human adults.1 Previously considered uncommon in companion animals, CVA is increasingly being recognized in dogs and cats as a result of advances in neuroimaging.2–7 Most types of CVA that are seen in humans have also been reported in dogs.3–7
The pathological classifications of CVA are divided into ischemic and hemorrhagic disease on the basis of the initiating insult to the nervous tissue.8 One form of hemorrhagic disease is intracranial SAH, which is characterized by a high mortality rate, increases in intracranial pressure, decreases in cerebral blood flow, and decreases in cerebral perfusion pressure.9 Intracranial SAH induces vasospasm that can result in cerebral infarction, which is characterized by a permanent ischemic tissue core surrounded by a potentially treatable penumbra.9
Recovery from a CVA in companion animals is probably more spectacular than in humans because nonhuman animals have a less prominent pyramidal system7,8; however, neurologic recovery after a CVA is ultimately dependent on the severity of the underlying infarction.10 Improvements have been made regarding the information on clinical recovery following a CVA and the neurobiological basis for that recovery. Evaluation of a CVA in nonhuman animals has provided cellular and molecular insights into events underlying clinical recovery and has been accompanied by the investigation of a wide range of therapeutic approaches.10
Concentrations of neuroexcitatory (glutamate, glycine, and aspartate) and inhibitory (GABA) amino acids after CVA have been investigated in humans and other animals.11–17 Glutamate, the predominant excitatory neurotransmitter, is found throughout the mammalian brain and participates in many metabolic pathways.18,19 However, glutamate is a potent neurotoxin and glutamate excitotoxicosis, mediated through NMDA receptors, has been implicated in the pathogenesis of many human neurologic diseases, such as ischemic stroke.11,13,14,20–22 The CSF concentrations of glutamate increase after a stroke, and it has been proposed that glutamate concentrations may serve as a means to assess the severity of a stroke.14
Currently, there is no specific treatment for ischemia resulting from a stroke. Treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (ie, statins), such as simvastatin, has provided protection for human patients at risk for major vascular events.1 There has been increasing interest in the use of simvastatin for the prevention of cerebral ischemia after SAH because it can ameliorate cerebral vasospasm and reduce neurologic deficits in mice23 and dogs.24 Statins can also attenuate inflammation induced by cerebral ischemia and have antioxidant properties, which may reduce excitotoxic effects.25 Statins have been reported to protect against NMDA-induced neuronal death.26–28 However, it is not known whether the positive effects of statins are associated with clinical alterations in neurotransmitter concentrations in the CNS.
Other strategies investigated for the treatment of ischemic stroke in humans include the use of immunosuppressive therapies.24 This has resulted in part from the fact that inflammatory by-products such as 20-hydroxyeicosatetraenoic acid, a potent vasoconstrictor, increase after SAH-induced stroke.29 Cyclosporine, a potent immunosuppressive drug, can ameliorate cerebral vasospasm associated with SAH in dogs.30 It is unknown whether reducing the inflammatory component associated with SAH is associated with alterations in amino acid neurotransmitter concentrations in the CSF. Because glutamate release is associated with inflammation,31 the use of immunosuppressive treatments (eg, cyclosporine) represents a potentially novel approach for the reduction of glutamate-mediated excitotoxicosis.
Investigating the concentrations of glutamate and GABA in the CSF after SAH may aid in understanding the role these neurotransmitters play in ischemia associated with SAH and alterations in their concentrations as a result of novel treatments. We hypothesized that dogs with SAH would have abnormalities in CSF concentrations of glutamate, aspartate, glycine, and GABA that would be attenuated after treatment with simvastatin and cyclosporine. The objectives of the study reported here were to investigate concentrations of glutamate, aspartate, glycine, and GABA in the CSF of dogs after SAH and the effects of treatment with simvastatin alone and simvastatin in combination with cyclosporine on concentrations of these neurotransmitters.
Materials and Methods
Sample—Samples of CSF were collected from 13 dogs as part of another study.24 No animals were used in the present study.
Induction of SAH and treatments—In the previous study,24 13 purpose-bred sexually intact female mixed-breed dogs from a research colony at the University of Missouri College of Veterinary Medicine were used. Dogs ranged from 7 to 18 months of age and each weighed approximately 20 kg. Dogs were assessed as clinically normal on the basis of results of physical and neurologic examinations, CBCs, serum biochemical analysis, urinalysis, and assessment of buccal mucosal bleeding times. Subarachnoid hemorrhage was induced in all dogs via a double SAH technique.30 Briefly, autologous blood (3 mL) was injected into the cerebellomedullary cistern, and a second 3-mL aliquot was injected 24 hours later. Dogs were assigned to 3 treatment groups. Five dogs received no treatment (group 1; control), 4 dogs were treated with simvastatina (20 mg/kg/d) and cyclosporineb (6 mg/kg/d; group 2), and 4 dogs received only simvastatina (20 mg/kg/d; group 3). Drugs were administered orally for 10 days beginning 6 hours after the second injection of autologous blood.
CSF collection and storage—Samples of CSF were acquired before the first injection of autologous blood (baseline; time 0), before the second injection of autologous blood, and on days 3, 7, and 10 (5 samples/dog). Cerebrospinal fluid was acquired by means of a standard technique. Dogs were positioned in right lateral recumbency, and a 22-gauge spinal needle was inserted into the cerebellomedullary cistern. Samples of CSF were collected by gravity flow. All CSF samples were immediately placed in polypropylene vials and stored at −80°C.
Measurement of amino acid concentrations in CSF—All CSF samples were evaluated at the University of Georgia College of Veterinary Medicine. Samples of CSF (100 μL) were deproteinized with methanol (100 μL) and centrifuged at 14,000 × g for 2 minutes in accordance with a method described elsewhere.32 After columns in a high-performance liquid chromatography systemc were derivatized with o-phthaldialdehyde and 3-mercaptopropionic acid, deproteinized samples were analyzed to determine concentrations of aspartate, glutamate, glycine, and GABA via high-performance liquid chromatography with fluorescence detection. Derivatized amino acids were resolved by binary gradient elution on a 3.5-μm, 150 × 3.0-mm amino acid analysis column.c The mobile phase comprised 40mM potassium phosphate (pH, 7.8) and a mixture of methanol, acetonitrile, and water (450:450:100 [vol/vol/vol]). The gradient was 0%, 0%, 35%, 60%, 100%, 100%, 0%, and 0% of the methanol-acetonitrile-water solvent at 0, 1.5, 10, 18, 20, 21, 23, and 28 minutes, respectively. Flow rate was 850 μL/min, and detection wavelengths were 235 nm (excitation) and 455 nm (emission). Derivatized samples (15 μL) were mixed with 15 μL of o-phthaldialdehyde reagent in the autosampler, and the reaction was allowed to proceed for 90 seconds prior to injection of the 30-μL volume.
Statistical analysis—All analyses were performed with statistical software.d Measured CSF variables were compared among treatments and time points by means of a repeated-measures teste to account for multiple observations within a dog over time. The repeated-measures model included factors for treatment, time, and the treatment-by-time interaction. Adjustments for multiple comparisons were made by use of the Tukey method. An unstructured covariance structure was used in all repeated-measures models. All hypothesis tests were 2-sided, and values were considered significant at α = 0.05. Simple correlation analysisf was used to test for correlations between the concentrations of the 4 neurotransmitters and the protein concentration, RBC count, and TNCC and between the RBC count and TNCC in the CSF.
Results
Aspartate, GABA, glutamate, and glycine concentrations as well as protein concentration, RBC counts, and WBC counts in CSF were summarized (Table 1). There were significant overall effects of treatment (P = 0.006) and time (P < 0.001) for glutamate concentrations (Figure 1). In the control group, the glutamate concentration significantly increased and reached the highest concentration by day 3 before returning to baseline values, whereas glycine and aspartate concentrations were not significantly altered from the baseline values at any time point. Glutamate concentrations on day 3 were significantly lower after treatment with simvastatin alone (P < 0.001) and simvastatin in combination with cyclosporine (P < 0.001), compared with the value for the control group. A significant (P = 0.008) increase in CSF concentrations of GABA was detected on day 3, compared with baseline concentrations, for all groups; however, there was no significant (P = 0.6) effect of treatment on these concentrations.
Mean ± SE concentrations of glutamate (A), aspartate (B), GABA (C), and glycine (D) in CSF acquired from 13 healthy purpose-bred research dogs in which SAH had been induced and that received no treatment (control [white bars]; n = 5) or were administered simvastatin alone (black bars; 4) or simvastatin in combination with cyclosporine (gray bars; 4). Dogs received 2 injections of autologous blood (3 mL/injection) into the cerebellomedullary cistern 24 hours apart (time 0 = sample collected immediately before the first injection of autologous blood). Drugs were administered orally for 10 days beginning 6 hours after the second injection of autologous blood. *Within a treatment group, the value differs significantly (P < 0.05) from the value for day 0.
Citation: American Journal of Veterinary Research 74, 8; 10.2460/ajvr.74.8.1111
Mean ± SD concentrations of neurotransmitters and protein and cell counts in CSF acquired at 5 time points from 13 healthy purpose-bred research dogs in which SAH had been induced and that received no treatment (control; n = 5) or that received simvastatin alone (4) or simvastatin in combination with cyclosporine (4).
| Variable | Day 0 | Day 1 | Day 3 | Day 7 | Day 10 |
|---|---|---|---|---|---|
| Aspartate (pg/mL) | |||||
| Control | 0.43 ± 0.04 | 0.43 ± 0.04 | 0.49 ± 0.04 | 0.41 ± 0.04 | 0.42 ± 0.04 |
| Cyclosporine and simvastatin | 0.32 ± 0.04 | 0.36 ± 0.04 | 0.40 ± 0.04 | 0.34 ± 0.04 | 0.34 ± 0.04 |
| Simvastatin alone | 0.37 ± 0.04 | 0.32 ± 0.04 | 0.34 ± 0.04 | 0.36 ± 0.04 | 0.37 ± 0.04 |
| GABA (pg/mL) | |||||
| Control | 0.22 ± 0.14 | 0.43 ± 0.14 | 0.75 ± 0.16a | 0.46 ± 0.14 | 0.41 ± 0.14 |
| Cyclosporine and simvastatin | 0.47 ± 0.14 | 0.48 ± 0.14 | 0.89 ± 0.14a | 0.24 ± 0.14 | 0.23 ± 0.14 |
| Simvastatin alone | 0.40 ± 0.14 | 0.35 ± 0.14 | 0.30 ± 0.14b | 0.31 ± 0.14 | 0.35 ± 0.14 |
| Glutamate (pg/mL) | |||||
| Control | 0.29 ± 0.10 | 0.85 ± 0.11 | 1.21 ± 0.11a | 0.55 ± 0.10 | 0.45 ± 0.10 |
| Cyclosporine and simvastatin | 0.33 ± 0.10 | 0.77 ± 0.10 | 0.43 ± 0.10b | 0.24 ± 0.10 | 0.24 ± 0.10 |
| Simvastatin alone | 0.34 ± 0.10 | 0.51 ± 0.10 | 0.40 ± 0.10b | 0.33 ± 0.10 | 0.33 ± 0.10 |
| Glycine (pg/mL) | |||||
| Control | 0.70 ± 0.40 | 0.80 ± 0.40 | 0.86 ± 0.46a | 0.66 ± 0.41 | 1.20 ± 0.41 |
| Cyclosporine and simvastatin | 1.60 ± 0.40 | 1.75 ± 0.40 | 3.61 ± 0.41b | 1.27 ± 0.41 | 1.29 ± 0.41 |
| Simvastatin alone | 0.80 ± 0.41 | 0.84 ± 0.41 | 0.85 ± 0.41a | 0.78 ± 0.41 | 0.80 ± 0.41 |
| Protein (mg/dL) | |||||
| Control | 30.5 ± 6.4 | 112.7 ± 6.4a | 38.6 ± 7.3 | 22.0 ± 6.4 | 20.7 ± 6.4 |
| Cyclosporine and simvastatin | 34.0 ± 6.4 | 80.4 ± 6.4b | 32.5 ± 6.4 | 24.0 ± 6.4 | 21.5 ± 6.4 |
| Simvastatin alone | 25.0 ± 6.4 | 55.0 ± 6.4c | 31.5 ± 6.4 | 20.0 ± 6.4 | 24.2 ± 6.4 |
| TNCC (cells/μL) | |||||
| Control | 2.5 ± 34.2 | 51.7 ± 34.2 | 224.7 ± 39.5a | 9.0 ± 34.2 | 6.2 ± 34.2 |
| Cyclosporine and simvastatin | 3.0 ± 34.2 | 76.5 ± 34.2 | 197.8 ± 34.2a | 21.0 ± 34.2 | 3.2 ± 34.2 |
| Simvastatin alone | 1.75 ± 34.2 | 53.2 ± 34.2 | 106.5 ± 34.2b | 5.0 ± 34.2 | 5.7 ± 34.2 |
| RBC count (RBCs/μL) | |||||
| Control | 1,255 ± 2,386 | 27,438 ± 2,386 | 5,142 ± 2,750 | 14 ± 2,386 | 18 ± 2,386 |
| Cyclosporine and simvastatin | 1 ± 2,386 | 35,826 ± 2,386 | 1,926 ± 2,386 | 95 ± 2,386 | 33 ± 2,386 |
| Simvastatin alone | 4 ± 2,386 | 32,299 ± 2,386 | 4,847 ± 2,386 | 4 ± 2,386 | 7 ± 2,386 |
Dogs received 2 injections of autologous blood (3 mL/injection) into the cerebellomedullary cistern 24 hours apart (time 0 = sample collected immediately before the first injection of autologous blood). Drugs were administered orally for 10 days beginning 6 hours after the second injection of autologous blood.
Within a column within a variable, values with different superscript letters differ significantly (P < 0.05).
Overall, there was a significant (P = 0.008) effect of treatment on the CSF concentrations of glycine. The glycine concentration was significantly (P < 0.001) increased, compared with that of the control group, after treatment with simvastatin and cyclosporine, especially on day 3.
The aspartate concentration did not differ significantly among treatments (P = 0.07) or time points (P = 0.37). There were significant correlations between protein and aspartate concentrations on days 1 (P = 0.039) and 3 (P = 0.011) and glutamate and protein concentrations on day 1 (P = 0.002) and glutamate concentration and TNCC on day 3 (P = 0.045).
Significant (P < 0.001) overall differences were detected among time points for protein concentration, RBC count, and TNCC. Specifically, the RBC count and protein concentration were significantly (P < 0.001) higher on day 1, compared with the values on day 0, and the TNCC was significantly (P < 0.001) higher on day 3, compared with the value on day 0. There were no overall significant effects of treatment on any of these variables; however, the TNCC on day 3 for the group treated with simvastatin alone was significantly (P = 0.03) lower than that in the control group. There were no significant correlations between RBC count and TNCC in the CSF, and there were no significant correlations between RBC count and concentrations of any of the CSF neurotransmitters at any time point.
Discussion
A stroke is a suddenly developing focal neurologic deficit resulting from a CVA.8 The causes of strokes can be divided into 2 basic groups: obstruction of blood vessels leading to ischemia and rupture of the blood vessel wall leading to hemorrhage.8,33 Intracranial SAH is a subset of the hemorrhagic form of CVAs, accounts for 5% of all strokes in humans, and is responsible for 25% of all fatalities related to strokes in humans.34 The prevalence of SAH in dogs is not known, but it is has been reported to be related to trauma, neoplasia, and myelographic examination.35,36 Regardless of its frequency in veterinary patients, it causes local vasospasm, which results in ischemic infarction, the most common form of CVA in dogs.7,33 The technique used to induce SAH in the dogs from which the CSF samples were obtained has been widely accepted as a standard method for investigations of vasospasm and cerebral ischemia.30 The onset of ischemia for this technique follows vasospasm and begins at approximately day 3 after SAH.24 This is important when interpreting changes in excitatory and inhibitory amino acids in the CSF. The mass effect associated with the SAH may also cause ischemic damage to brain parenchyma, which would have an earlier time course.
Analysis of results of the present study confirmed clinical and experimental findings that CSF glutamate concentrations are increased by day 3 after intracranial SAH and ischemic brain damage.12,13,37,38 Investigators of 1 report39 determined that microdialysis concentrations of glutamate were 300 times as high as the reference range for several days after stroke onset in the cortex of a human patient with a massive cerebral infarct. This is in contrast to the increase in glutamate concentrations for in vivo experimentally induced focal ischemia, which is transient and lasts from only 1 to 2 hours to 48 to 72 hours.20,22,40,41 The difference is believed to be related to both the onset and the progression of the ischemic process because in human patients with stable ischemic stroke, glutamate concentrations returned to reference range values within 6 hours after stroke onset, which was not the case for humans with progressive ischemic stroke.13,14
The CSF concentration of glutamate during experimentally induced ischemia correlates positively with infarct size and has been proposed as a good marker for severity of the underlying disease.41–43 The accumulation of glutamate in extracellular spaces is attributable to an increase in release as well as a decrease in reuptake.44 Both alterations are secondary to cerebral ischemia, which is responsible for the energy failure of the ion exchange system and for depolarization of membranes.45 On the basis of results of the study reported here, glutamate concentrations in the CSF may be, at least in part, a useful marker of injury to the brain within a specific time period, although a correlation with severity of the injury was not investigated.
Glutamate toxicosis may have a major role in the progression of ischemic disease in the brain.42,43,46,47 This is based on the neurotoxic effect of glutamate within the penumbra or peripheral zone of an ischemic lesion causing an increase in the extent of a cerebral injury.40,48 Because of this, medications that may reduce the concentrations of glutamate, such as NMDA-receptor inhibitors, have been suggested as potential treatments for CVA, even at a relatively late stage of disease.49 The possibility that excessive release of excitatory amino acids results in exacerbation of neuronal damage has generated great interest. The major assumption of this postulation is that the release of excessive amounts of neurotransmitters, as exemplified by glutamate, follows a variety of insults, including trauma, and that excessive accumulation of excitatory amino acids initiates a complex process of cellular injury, which if uninterrupted, will result in calcium influx and cell death.31,50,51 Excitotoxicosis is triggered by the excessive release of glutamate from presynaptic nerve terminals and astrocytes into the extracellular space, with consequent overstimulation of glutamate receptors, especially NMDA receptors.51,52 This receptor overstimulation leads to excessive influx of calcium ions (and sodium ions) through glutamate receptor–gated ion channels, which is followed passively by movements of chloride ions and water.50 The resulting combination of intracellular volume and overload of calcium ions induces various lethal metabolic derangements, internal swelling of organelles, and failure of plasma membranes, which results in necrosis.53
Concentrations of the excitatory neurotransmitter aspartate were not increased in the CSF after the experimentally induced condition in the dogs of the present study. This finding is in agreement with results for other techniques for experimentally mimicking stroke and is in accordance with evidence that suggests aspartate is not associated with the excitotoxic theory of stroke damage.14
Increased glycine concentrations in the CSF have been measured in subjects in which other methods have been used to experimentally induce progressive ischemic stroke,14,41 but we did not detect increases in glycine concentrations in the present study. However, the result for the present study is similar to findings in humans with ischemic lacunar strokes, which has given rise to the theory that high glycine concentrations may be detected only when there are extremely large ischemic lesions.17 It has been purported that even though glycine is predominantly an inhibitory neurotransmitter in the spinal cord, it is needed in the brain for glutamate-induced activation of the NMDA channel and could exacerbate the harmful effects of increased glutamate concentrations.54 This is of concern because of the fact that treatment with simvastatin in combination with cyclosporine significantly increased glycine concentrations on day 3, compared with concentrations in the control dogs. The reason for this is unclear. However, considering that concentrations of endogenous glycine typically are sufficiently high enough to saturate binding sites of the brain's glutamate receptors, an increase in glycine release would not necessarily cause higher activation of NMDA receptors, although further investigations are warranted.14,55
Neurotransmission of GABA results in increased chloride flux across the postsynaptic membrane and hyperpolarization.17 These actions counterbalance the toxic effects of glutamate during cerebral ischemia, which is supported by the neuroprotection of the GABAA-receptor agonists in animals with experimentally induced stroke.17 A sustained increase in GABA outflow in an ischemic brain has been detected via microdialysis after permanent occlusion of the middle cerebral artery in rats.56 It is hypothesized that such an increase serves to protect the brain from injury.57 An increase in the GABA concentrations in the CSF was detected parallel with the increase in glutamate concentrations on day 3 of the present study, which is similar to the result for rats with experimentally induced ischemia.56 Other experiments17 have revealed a reduction in CSF concentrations of GABA in humans and nonhuman animals with acute ischemic stroke, so the validity of the results of the present study must be confirmed.
The use of statins, such as simvastatin, for the treatment of vasospasm and cerebral ischemia and its associated neurologic deficits has a strong scientific rationale23 and has shown promise in mice23 and dogs24 with experimentally induced CVA and humans in phase II clinical trials.55–60 Statins can increase expression of endothelial nitric oxide synthase, enlarge the diameter of spastic arteries, and increase cerebral blood flow.23 Furthermore, statins attenuate inflammation induced by cerebral ischemia, decrease platelet activation, enhance fibrinolysis, and have antioxidant properties.25 Such pharmacological properties have created interest in the potential use of these drugs to reduce glutamate-mediated excitotoxicosis in subjects with cerebral ischemia. Statins reportedly protect against NMDA-induced neuronal death, but the mechanism underlying this protection remains unclear.26–28
On the basis of experimental and clinical results, we decided to evaluate the use of simvastatin on neurotransmitter concentrations in the CSF. When used alone, simvastatin was associated with significant reductions of glutamate concentrations in the CSF on day 3 but did not have any effect on the other neurotransmitters. This result suggested that simvastatin may have a role in reducing glutamate-mediated excitotoxicosis associated with CVA by reducing extracellular glutamate concentrations through an unknown mechanism. The dose of simvastatin was chosen on the basis of doses used in previous studies61,62 conducted to determine its metabolism and a lack of toxic adverse effects in dogs. To our knowledge, dose-response studies have not been performed with simvastatin and would be necessary to establish the most efficacious treatment regimen for dogs with CVA.
Cyclosporine is a short polypeptide that blocks the activation of lymphocytes and other immune system cells.63 Immunosuppressants such as cyclosporine exert neuroprotective and neurotrophic action in animals with traumatic brain injury and sciatic nerve injury as well as focal and global ischemia.63,64 Additionally, cyclosporine can ameliorate cerebral vasospasm associated with SAH in dogs.30 Given that glutamate release is associated with inflammation,31 we wanted to investigate the effect of cyclosporine treatment on glutamate concentrations in the CSF. Because the present study represented an extension of a previous study24 conducted to investigate the additive effect of cyclosporine and simvastatin on vasospasm, it was not possible to evaluate the sole effect of cyclosporine on neurotransmitter concentrations. When combined with simvastatin, there continued to be a significantly lower glutamate concentration in the CSF on day 3, compared with the concentration in the control dogs, but there was no significant difference between the 2 treatment groups, which suggested a lack of additive effect for the cyclosporine treatment. The combination treatment also significantly increased glycine concentrations in the CSF on day 3 from that for the control dogs, but it had no effects on the GABA or aspartate concentrations. On the basis of the results of this study, cyclosporine did not have any potential beneficial effect on glutamate-mediated excitotoxicosis. However, the dose used in this study was selected on the basis of anti-inflammatory doses reported for SAH in dogs,65 and other dose regimens may have different effects on neurotransmitter concentrations.
Plasma samples were not evaluated in the present study, but a significant correlation between CSF and plasma glutamate concentrations has been reported in previous CVA studies.14,66,67 It was suggested in those studies that plasma glutamate concentrations, if elevated, may increase glutamate concentrations in the CSF. Considering that increases in plasma concentrations of glutamate have only been reported in association with cirrhosis, advanced carcinoma, and HIV infection, it is not thought that the increases in glutamate concentrations in the CSF of the healthy dogs in the present study represented an increase attributable to high concentrations of glutamate in the plasma. This is also supported by the lack of association between glutamate concentrations in the CSF and blood contamination of the CSF. Blood contamination of the CSF was unavoidable because it was related to the double-hemorrhage induction technique.
We believe that concentrations of glutamate in the CSF of dogs may be a useful marker of ischemic brain injury. Simvastatin administration can ameliorate high glutamate concentrations, which offers a potential cerebroprotective role and warrants further investigation.
ABBREVIATIONS
| CVA | Cerebrovascular accident |
| GABA | γ-Aminobutyric acid |
| NMDA | N-methyl-d-aspartate |
| SAH | Subarachnoid hemorrhage |
| TNCC | Total nucleated cell count |
Zocor, Merck, Whitehouse Station, NJ.
Cyclosporine, Pliva Inc, Zagreb, Croatia.
Zorbax Eclipse, Agilent Technologies, Santa Clara, Calif.
SAS, version 9.1, SAS Institute Inc, Cary, NC.
Proc Mixed, SAS, version 9.1, SAS Institute Inc, Cary, NC.
Proc Corr, SAS, version 9.1, SAS Institute Inc, Cary, NC.
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