New Magnetic Resonance Spectroscopy Biomarker for Monitoring Neurodegenerative Diseases: Animal Models

Key words: Creatine kinase/Saturation transfer 31P NMR/Chronic cerebral hypoperfusion/Neurodegenerative diseases/ Animal model Creatine kinase (CK) plays a central role in energy transfer in cells with high-energy demands, and the enzyme is rather susceptible to oxidative inactivation. The aim of the present study was to investigate whether the rate constant of forward CK reaction (k for) is a suitable indicator of alterations in cerebral energy metabolism. We monitored k for in the rat brain non-invasively by in vivo phosphorus (31 P) magnetic resonance spectroscopy (MRS). To alter energy metabolism , we applied following experimental models: Huntington's disease, diabetes mellitus, chronic alcohol intoxication and chronic cerebral hypoperfusion (vascular dementia model). Results of our 31 P MRS experiment confirm importance of creatine kinase/phosphocreatinine (CK/PCr) system in the regulation of brain energy metabolism in vivo because a kinetic parameter k for was significantly changed in all above animal models that simulate neurodegenerative diseases or commonly during oxidative stress. Using this method we distinguished vascular dementia (VD) and Huntington disease (HD), because in VD model a kinetic parameter k for decreased and in the case HD increased. Considering the importance of CK for the maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which various neurodegenerative diseases might be monitored just by means saturation transfer method 31 P MRS.


INTRODUCTION
Metabolism of adenosine triphosphate (ATP) is key process for cell live and involves ATP synthesis and its utilization for supporting brain function and activation.The ATP metabolism is tightly coupled to the phosphocreatinine (PCr) metabolism via the enzyme system of creatine kinase.CK plays a central role in energy transfer in cells with high and fluctuating energy requirements and it is very susceptible to oxidative stress [1][2][3][4] .It would be valuable to establish a noninvasive approach for studying this chemical process in the animal (human) brain in vivo.Rate constants of CK reaction can be investigated by magnetization transfer in vivo 31 P MRS experiment 5,6 .By means of this technique we studied reaction kinetics of reversible exchange of the phosphate group in the reaction catalyzed by CK PCr 2-+ MgADP -+ H + MgATP 2-+ Cr (1)   in the adult and aged rat brains in various chronic models of neurodegenerative diseases [7][8][9][10][11] .
The forward rate constant of CK, k for (PCr =>ATP) was studied in adult and aged rat brains in following models of diseases: i) Huntington's disease (HD), ii) diabetes mellitus (DM), iii) alcohol intoxication and iv) vascular form dementia (VD) of Alzheimer's disease modeled by chronic cerebral hypoperfusion.The aim of these experiments was to demonstrate that in chronic pathological states the pseudo first-order rate constant k for of the CK forward reaction is a better indicator of changes in the brain energy metabolism than the conventional steadystate in vivo 31 P MRS.

Animal models
Wistar rats with the age 3-10 (adult) and 14-24 (aged) month were used in 31 P MRS experiments.
Chronic alcohol intoxication was modeled by administration of 30% ethanol (3 ml/day, intragastrically) to 3 months old female rats for the period of 3 weeks.Control group (C) received water as a vehicle 7,8 .
Vascular dementia (VD) model was prepared by a minimally invasive surgical technique of 3-vascular occlusion by brachiocephalic trunk, which eliminated simultaneously right common carotid artery and right vertebral artery 9,12 .6-10 months old (adult) and 14-17 months old (aged) male Wistar rats were used in this experiment.Saturation transfer in vivo 31 P MRS were performed two and ten weeks after vascular occlusion 8,9 .

In vivo 31 P magnetic resonance spectroscopy
In vivo 31 P MRS experiments were performed at 4.7 T on SISCO 200/300 imaging spectrometer equipped with horizontal bore magnet for measurements on animals. 31P MR spectra were collected using 16 mm surface coil.The static magnetic field was shimmed using the proton signal of water that showed a typical line width of 20-35 Hz.Relative concentrations of phosphate metabolites were determined from integrals of their signals in 31 P MR spectra using program MESTRE-C 1.5.1.Time-dependent 31 P MRS saturation transfer was applied to determine the pseudo-first order rate constant of forward CK reaction (k for ) as described previously 6,9,13,14 .Time dependent saturation transfer allows one to measure simultaneously two parameters, T 1 and k for .Saturation of the γ-ATP resonance for increasing time periods induces an exponential decay of the PCr resonance to a new steady state (Fig. 1).
The saturation was accomplished by the DANTE pulse sequence.The k for was calculated according to the equation 9,13,14 where M 0(PCr) and M* (PCr) are the PCr signal intensities in the reference spectrum and in the spectrum after 10second irradiation of the γ-ATP resonance, respectively.T 1app(PCr) is the apparent longitudinal relaxation time of the PCr signal in the presence of the γ-ΑTP saturation.
To check the validity of the results, the T 1PCr values were calculated using the following equation 9, 13, 14 Calculated values k for and T 1PCr were expressed as means ± standard deviations.The Student's t tests were performed to compare differences between groups.

RESULTS AND DISCUSSION
Our findings suggest that the CK reaction could play a key role in energetic system of adult and aged brains in above models of neurodegenerative diseases.While the analysis of steady-state 31 P MR spectra revealed significant alteration in brain PCr/βATP ratio only in diabetic group, we found significant changes of brain k for in all chronic models of neurodegenerative diseases.Compared to controls k for increased in HD and DM models and decreased in chronic alcohol intoxication and VD models (Table 1).
From kinetic 31 P MRS spectra we found in case of HD small (+13.4 %), but statistically significant an increase in k for (Table 1).Exact mechanism responsible for the increase of the pseudo-first-order rate constant for the CK reaction k for is presently not clear.However, in vivo 31 P MRS transfer measurements revealed a correlation between CK flux and brain 2-deoxyglucose uptake 5,6 .Our result of increased activity of CK corresponds well with reported increased 2-deoxyglucose uptake in basal ganglia of HD patients 1,5 .
Similar increase of k for (+22.9 %) we obtained in the model of diabetes mellitus in which also increased brain PCr/βATP ratio was found (Table 1).In both above mentioned models the function of respiratory chain in brain mitochondria was diminished 11 .We suppose that decreased brain mitochondrial ATP production could be partly compensated in a certain stage of these pathological states by increased activity of CK system.The fact that the increase of k for in these models of neurodegenerative diseases could be prevented by application of CoQ 10 provides evidence that k for reflecting activity of CK system is sensitive marker of changes in brain energy metabolism 10,11 .The length of the saturation period is given below each spectrum.In the control experiment, the saturation was off-resonance.The saturated γATP signal is labelled with asterisk.
S. Kašparová, Z. Sumbalová, J. Horecký, P. Bystrický, V. Mlynárik, A. Gvozdjáková, T. Liptaj Long-term consumption of alcohol can lead to brain atrophy and neurological dysfunction.Ethanol and its metabolite acetaldehyde are directly neurotoxic and its acute and chronic intoxication with ethanol induces oxidative stress in CNS and peripheral nerves 7 .The conventional 31 P MRS revealed statistically significant decrease of intracellular pH in the brain of rats treated by ethanol, but it did not show any significant variation in the content of high-energy phosphates after 14 weeks of drinking the high ethanol doses 7 .However, measurement of fluxes in the direction from PCr to ATP CK reaction showed significant decrease of the forward CK rate constant in alcoholic rat brain 2,8 (Table 1).
The study of cerebral hypoperfusion in rats is great importance for a better understanding of cerebrovascular dynamics in various neurodegenerative diseases including Alzheimer's disease (AD).The characteristic pathology of AD involves microvascular degeneration and chronic cerebrova scular insufficiency 12 .Two factors must be present before cognitive dysfunction and neurodegeneration is expressed in AD brain: advanced aging and presence of a condition that lowers cerebral perfusion 12 .Compared to the control group of healthy aged rats the hypoperfused rats showed statistically significant decrease in k for both 2 and 10 weeks after occlusion (Table 1).It should be noted here that adult and aged rats kept for 10 weeks under conditions of severe hypoperfusion, showed no statistically significant changes in conventional in vivo 31 P MRS spectrum.Dysfunction of the CK system under AD conditions has also recently been reported 4,15 .The causes of decreased levels of cytosolic brain CK isoform (BB-CK) in postmortem brain in mental pathology are subject of discussions 15 .As it was recently demonstrated, BB-CK energy transfer in the brain is important for habituation and spatial learning behaviour 16 .Dynamic study of 31 P MR spectroscopy revealed strong reduction of PCr New magnetic resonance spectroscopy biomarker for monitoring neurodegenerative diseases: animal models =>ATP phosphorus flux in mice with ablation of cytosolic BB-CK, while ATP and PCr levels were unaffected in these mice 16 .Thus, the rate constant of CK, k for measured in the brain reflects changes of BB-CK activity 8,9,16 .
We confirmed that a metabolic capacity of the creatine kinase reaction in the brain in vivo was significantly changed in various animal models simulating neurodegenerative diseases, such as AD, HD or generally during oxidative stress.
We have concluded that the investigation of the kinetic parameters using in vivo 31 P MRS magnetization transfer method contributed to a better understanding of the underlying processes in various neurological disorders.Using this method we distinguished AD and HD, because k for decreased in AD model while it increased in HD.This technique can be used as a noninvasive in vivo biomarker for age-related neurodegenerative diseases as it can reveal energy metabolism impairment of brain tissue, which is not yet detectable by conventional MRS methods.

Fig. 1 .
Fig. 1.Time dependent 31 P NMR saturation transfer experiments.Series of saturation experiments with variable time of irradiation (0.3-10 s) of γATP signal.The length of the saturation period is given below each spectrum.In the control experiment, the saturation was off-resonance.The saturated γATP signal is labelled with asterisk.

Table 1 .
Parameters determined by 31 P MRS methods in the rat brain in models of neurodegenerative diseases n -number of animals per group; k for -the pseudo-first order rate constant of forward CK reaction determined in brain in vivo by 31 P MRS saturation transfer; T 1 (PCr) -longitudinal relaxation time of phosphocreatine in absent γ-ATP saturation, a control parameter; PCr/βATP ratio calculated from in vivo 31 P MR spectra of the rat brain.C -control rats; *p < 0.05, ** p < 0.01 vs. control group, # # P < 0.001 vs. DM group, a P < 0.05 vs. control adult rats