Usefulness of combined pseudo-continuous arterial spin labelling and spectroscopic analysis in schizophrenic Egyptian population sample

Background Schizophrenia is a prevalent psychiatric disorder that affects 1% of the global population. Schizophrenia frequently begins in late adolescence or early adulthood, causing significant educational, social, and economic costs for people and society. Functional neuroimaging research on schizophrenia physiopathology has been beneficial. Arterial spin labelling (ASL) is one of functional magnetic resonance imaging (fMRI) technologies that assess brain function without radiation. ASL uses magnetic resonance (MR) imaging to quantify tissue-level brain perfusion non-invasively. Arterial spin labelling (ASL) is one of the functional magnetic resonance imaging (fMRI) technologies that assess the brain function without radiation hazards. ASL uses magnetic resonance (MR) imaging to quantify tissue-level brain perfusion non-invasively. Many publications were performed on role of different advanced MRI techniques in schizophrenia,


Background
Schizophrenia is a prevalent psychiatric disorder.It is one of the world's 10 leading causes of disability assessed by DALYs scale and, despite current progress in research and treatment, continues to be a major aetiologic, diagnostic, and therapeutic challenge.It has been estimated that around 1% of the world population suffers from this chronic and disabling disorder [1].Functional neuroimaging studies have proved to be an invaluable source of information about the physiopathology of schizophrenia [2].
Regional cerebral blood flow (rCBF) is defined as the volume of blood delivered to a given mass of brain tissue over a given time [3].Many previous studies have demonstrated that schizophrenia patients exhibited an increased or decreased resting-state CBF by SPECT in multiple brain regions, especially a decreased CBF in the prefrontal cortex.However, this technique is quite invasive, with a long acquisition time and low spatial resolution.In contrast, the arterial spin labelling (ASL) MRI is a non-invasive technique that can rapidly quantify CBF [4].
Arterial spin labelling (ASL), on the other hand, is based on the quantification of the water flow within brain regions, which is considered to provide an indirect measure of metabolism.Arterial spin labelling (ASL) is a magnetic resonance (MR) imaging technique that enables the measurement of brain perfusion non-invasively at the tissue level.Benefiting from the contrast of inflowing magnetically labelled blood, ASL obviates an exogenous contrast agent [5].
In arterial spin labelling, the tracer is a magnetic label (tag) applied to water molecules of flowing blood prior to their reaching the region under assessment [6].Such labelling consists in an inversion of proton spins of arterial water.After an interval that allows water molecules to be exchanged within tissues, an image, referred to as 'labelled' or 'tagged' , is acquired.In this image, blood water is in a different magnetization state from that of static tissue water.Also, a control image of the same slice is acquired in which inflowing blood is not labelled.By calculating the difference between control and tagged images, investigators can create an image that corresponds to the proportional rCBF in a given brain region [7].This technique has been used to explore CBF alterations in schizophrenia and has demonstrated increased CBF in the striatum, decreased CBF in the prefrontal and anterior cingulate cortices, and increased or reduced CBF in the thalamus and temporal cortex.It is not known if these changes in rCBF are due to genetic influences, but findings of altered rCBF in first degree relatives of patients with schizophrenia indicate a familial and possibly genetic influence [8,9].

Aim of the work
The aim of this study was to assess the capability of combined use of ASL and multi-voxel MRS in early detection and follow-up of schizophrenia by evaluation of the changes in ratios of neuronal metabolites by MRS and altered rCBF in different brain regions, compared to healthy individuals.

Study population
The current study design was prospective (case-control study), that was carried out on two groups: First group was 30 patients who were suspected clinically as schizophrenic patients, and the second group was 20 healthy volunteers as a control group for comparison.Those patients were referred to Diagnostic Radiology and Medical Imaging department from Neuropsychiatry department of our institute, over a period of 12 months, starting from August 2021 till July 2022.The study was performed after approval by the Research Ethical Committee.
The inclusion criteria of the studied cases were: patients who were clinically diagnosed as schizophrenic patient for the first time or stopped treatment more than 3 months.Their ages are between 20 and 60 years old.Both sexes were included in the study.Healthy volunteers of matched 20 patients (12 Males and 8 Females) were selected randomly as a control group to match the gender and aged of diseased group.While patients who are contraindicated to go under the magnet as patients with cardiac pacemakers, non-compatible intracranial clips of brain aneurysms and intraocular metallic foreign body were excluded from our study, as well as schizophrenic patients who were under medical medication (within 3 months from the study time).Also, claustrophobic people or patients with previous neurological disorders or traumatic brain injuries were excluded from the present study.

The present study was divided into 2 groups
Group 1 Thirty schizophrenic patients: 20 males and 10 females aged from 20 to 50 (12 of them were newly to measure cerebral blood flow and identify decreased rCBF without any contrast administration, and it could be repeatable which helps in early diagnosis as well as follow-up of the progression of the disease. diagnosed as schizophrenic who met the DSM 5 criteria [10], another 18 chronic schizophrenic patients and stopped their medical treatment for more than 3 months).
Group 2 Twenty age and sex matched healthy volunteers were enrolled in the current study as control group they were 12 males and 8 females with their ages ranged from 20 to 50 years they had no medical or neuropsychiatric illness.
All patients were subjected to the following:

Full history
Proper history taking:-Personal history: as regard name, age and sex.Present history: as regard the presenting symptoms as positive symptoms, negative symptoms, and cognitive dysfunction.Past history: as regard any previous neurological diseases, trauma, intracranial haemorrhage, CNS infection, or previous operations, and family history of any CNS anomalies.

Clinical examination
Psychiatric Assessment using the psychiatric clinical sheet in Neuropsychiatry department.Clinical diagnosis of schizophrenic cases was done using Mini International Neuropsychiatric Interview (MINI) [10], which is a short, comprehensive feasible, reliable, and valid systemic diagnostic psychiatric interview, with short administration time.Assessment of Schizophrenia severity using Positive and Negative Syndrome Scale (PANSS) [11].The Positive and Negative Syndrome Scale was used for assessment of psychosis.The PANSS is a medical scale used for measuring symptom severity of patients with schizophrenia and was published in 1987.The scale is a 30-item, seven-point rating instrument.Of the 30 parameters assessed, seven were chosen to constitute a Positive Scale (score range 7-49), seven a Negative Scale (7-49) and the remaining 16 a General Psychopathology Scale .

All included individuals were subjected to MRI study
General Electric (GE) 1.5 Tesla (closed magnet) MR machine with a standard circularly polarized head coil was used to perform all MRI scans.Using a standard eight channels' head coil, head support pillow to minimize patient's movement.
The patient lied supine during the exam in comfortable position during the MR exam to minimize his motion.The head was introduced first.The examination time ranged from 15 to 30 min.The slice thickness was 4 mm, the matrix was 256 × 256, and the field of view was 220-240 mm.No sedation was given.

Patient preparation
All metallic objects like pins and earrings were removed and emptied the bladder prior to examination (to prevent patient irritability during examination).
Post-processing of MRS was performed using MR workstation software (ADW 4.7 Vantage, GE Medical Systems), regions of interest (ROI) were placed in both frontal gyri (especially right medial frontal gyrus), medial cingulate gyri.Parieto-temporal cortices, hippocampi, thalami, basal ganglia, cerebellum).The peak ratios were calculated from the integration of the single peak including: NAA/Cr, NAA/Cho, and Cho/Cr.Interpretation of the spectral peaks was done quantitatively by three experienced radiologists (25, 11, and 3 years).This quantitative assessment represents a relative concentration of the metabolites using the integral ratios and comparing it to the control group.

Arterial spin labelling technique
The label was created by applying radiofrequency pulses to invert the bulk magnetization of the blood water protons.Images were acquired after the labelling and inflow period by using rapid acquisition techniques such as echo-planar imaging, gradient-and spin-echo imaging (GRASE), or three-dimensional fast spin-echo imaging using a stack-of-spirals approach.
During ASL image acquisition, repeated label and control images are typically interleaved.Perfusion contrast was obtained by pair-wise subtraction of the label and control acquisitions.
Post-processing of ASL was performed on GE workstation software, qualitative assessment of colour map, quantitative analysis was done, and absolute regional cerebral blood flow (rCBF) was estimated in the scanned hypo-perfused areas, by modelling expected signal changes in the brain.
The labelling is continuous through a thin slice at the neck level.The inversion of the magnetization is obtained by the joint application of continuous pulsed radiofrequency for 2-4 seconds and a magnetic field gradient in the direction of the flow.
Tagging efficiency of CASL may be affected by variations in flow velocity, which makes the average inversion efficiency of CASL (80-95%) lower than PASL (95%).However, the closer inversion to the imaging plane minimizes the loss of perfusion signal caused by T1 relaxation and somehow compensates for the lower inversion efficiency.

Statistical analysis
Data were analysed using the IBM ® SPSS statistical software, version 21.We used the one-sample Kolmogorov-Smirnov test to check the normality of data and the data were parametric (normally distributed).Numerical data were presented as mean and standard deviation (SD), and categorical data were presented as number and percentage.
Chi-squared test was used for comparing the qualitative one-way of variance (ANOVA) test to compare the means in different groups.Post hoc test (LSD) was done to determine the significance between each two groups.
Linear correlation analysis was done by Spearman coefficient correlation and used for testing the positive or negative associations between different variables.The level of significant was adopted at p<0.05.
Sensitivity The capacity of the test to correctly identify diseased individuals in a population "true positive".The greater the sensitivity, the smaller the number of unidentified cases "false negative".
Specificity The capacity of the test to correctly exclude individuals who are free of the disease "true negative".The greater the specificity, the fewer "false positive" will be included.
Positive Predictive value (PPV) The probability of the disease being present, among those with positive diagnostic test results.

Negative Predictive value (NPV)
The probability that the disease was absent, among those whose diagnostic test results were negative.
Accuracy Rate of Agreement Intra-class Correlation coefficient It was used for the agreement between each two observers.The intra-class correlation coefficient (ICCs) were classified using a system suggested by Koo and Li (2016) as follows: less than 0.50 Z poor agreement; 0.50 to less than 0.75 Z moderate agreement; 0.75 to less than 0.90 Z good agreement; and 0.90 or greater Z high agreement.If a P value was less than 0.05 , this was considered a statistically significant data.

Results
This prospective case-control study was carried out in the period from 1st August 2021 till July 2022, the current study enrolled 30 schizophrenic patients and 20 ageand sex-matched healthy volunteers as a control group.The mean age in the patient group was 35.6 + 4.5 years, and in the control group, it was 31.1+3.8 years.Males constituted 66.7% of the patients' group (20 patients) and 60.0% of the control group (12 participants).Nineteen patients were educated and only 10 patients of the schizophrenic cases were employed representing (33.3% of cases).Concerning the marital status 18 patients are single.As for special habits 16 patients were smokers as described in table 1.
Regarding family history 16 out of 30 patients had positive family history of psychiatric illness who were 53.3% of cases, meanwhile the rest of the cases had no positive family history of any psychiatric disorder.
According to the patients' symptomatology data, majority of cases (93.3% and 90%) had delusions and hallucinations, respectively.
Regarding the clinical tests which were performed in the current study, as follows: correct response on Wisconsin Card Sorting Test (WCST) was significantly lower among both groups A and group B than control and group A had lower performance than group B with mean 31.47 ± 7.49 for group (A), 39.97 ± 8.17 for group (B), and 49.83 ± 5.36 for control patients.While both groups A and B had significantly higher errors than control, group A had significantly higher errors than group B with mean 31.27 ± 5.39 for group (A), 25 Concerning MR spectroscopic analysis in the studied patients' group, it demonstrated NAA/Creatine ratios which had statistically significant difference between both groups at hippocampi and thalami (P.value = 0.06* and 0.08*), respectively.As well as, concerning NAA/ Choline ratios, there was also significance at hippocampi and thalami, (P value = 0.04* and 0.06*) respectively.Moreover, regarding choline/creatine ratios, the lowest ratios were appreciated at cingulate gyrus region in both schizophrenic subgroups, with no statistically significant difference between healthy control group and patients in all scanned regions of interest as shown in Tables 3, 4, 5.
Assessment of the diagnostic performance of the two studied imaging modalities revealed that the highest area under the curve (AUC) was for ASL in inferior frontal gyrus (0.943) that showed sensitivity of 90.0% and specificity of 95.0% with a cut-of value of ≤ 44, medial frontal gyrus AUC was (0.913) with sensitivity of 93.33% and specificity of 85.0%, the parietal lobe AUC was (0.894) with sensitivity of 83.33% and specificity of 90.0%, while the anterior cingulate gyrus showed AUC of (0.876) with sensitivity and specificity of 76.67% and 90.0%, respectively, as mentioned in Fig. 1, Table 6.Comparison between the three studied observers according to different parameters is shown in

Discussion
Schizophrenia spectrum and other psychotic disorders affect up to 3% of the population, are accompanied by prominent social and occupational functional impairments, and represent one of the leading causes of disability worldwide.Psychotic disorders have been suspected to be associated with disturbances in the cerebral blood flow (CBF) [12].
The two most common perfusion MRI methods are dynamic susceptibility contrast (DSC) and arterial spin labelling (ASL) [13].
Arterial spin labelling (ASL) imaging has been validated extensively against other perfusion methods by demonstrating accurate and reproducible quantification of CBF.Since it is completely non-invasive, ASL imaging has become useful for many clinical and research applications [12].
The aim of this work was to evaluate the combined added value of p-CASL-perfusion MRI and MR spectroscopy (MRS) in schizophrenia.In the present study, 30 patients (20 males and 10 females aged from 20 to 50 years old) who were known to be schizophrenic underwent conventional brain MRI, together with ASL-MRI and MRS.Also, a matched group of 20 healthy volunteers (12 males and 8 females aged from 20 to 50 years old) were subjected to the same MRI examinations.
In this study, more than 66.7% of patients were males.This matches with the study of Nawka et al. [14] who found by clinical observation that males are more affected than females with earlier onset by (3-5) years.
A wide range of age groups were included in this study, ranging from 20 to 48 years, with most patients in the schizophrenic group (60%) were at the age ranged from 20 to 30, and the mean age was 35.6 ± 4.5 years.This agrees with Nawka et al. [14] who provided robust, global epidemiological estimates of age at onset for mental disorders, including schizophrenia, through a systematic review with meta-analysis, and found that the peak age of patients with schizophrenia was 33 years.In addition, according to the Global Burden of Disease Study 2016 that was published by Charlson et al. [15] and demonstrated the global epidemiology and burden of schizophrenia, as with prevalence, the peak disease burden was observed at around 30-40 years of age, where individuals are most likely to be economically productive.
The current study showed that about half of the patients (46.7%) had a positive family history of schizophrenia.This is in line with what has been reported that schizophrenia is a highly heritable    disorder according to the study made by Hilker et al. [16] who showed in their study that a substantial genetic component in liability to schizophrenia, with a heritability estimate of 79%.All patients underwent conventional brain MRI to evaluate brain volume and any structural abnormalities, and it was noted that there is significant difference in the whole brain, thalamus, frontal, and temporal lobe volumes between the schizophrenia patients and the control group.These results are congruent with previous study of brain MRI by Guo et al. [17] who investigated schizophrenia individuals and controls with MRI brain and found that schizophrenia cases exhibited greater progressive brain reductions than controls, mainly in the frontal and temporal lobes.
In the current study, significant rCBF decrease in ACC, parietal and frontal lobes and increase putamen and hippocampus were found.In agreement with the current findings, Guimarães et al. [18] reported decreased rCBF in the frontal lobe of patients with schizophrenia.Convergent results of decreased rCBF in the ACC are consistent with clinical observations and other imaging studies.These results were in line with Zhu et al. [19] who identified decreased CBF in the left medial frontal gyrus, a cortical region of the frontal lobe and identified decreased CBF in the anterior cingulate cortex as well.
The striatum is studied in schizophrenia by Howes and McCutcheon [20] who reported higher flow in schizophrenia patients.
The present work implemented the use of MRS for assessment of patients with schizophrenia and the control healthy subjects.We found that NAA concentration in anterior cingulate cortex, thalami, basal Ganglia and hippocampus is significantly lower than in healthy controls, this was in agreement with the findings of Szulc et al. [21] study.
Glutamate was decreased in ACC and left superior temporal gyrus in both chronic cases and first attacks by same ratio.In basal ganglia and thalami, it was increased in 1st diagnosed cases rather than chronically ill patients.This goes with the study done by Poels et al. [22], who reported higher levels of glutamine in the ACC in 21 antipsychotic-naïve schizophrenia subjects than levels in 21 chronic, medicated schizophrenia subjects.
In the present study, there was significant reduction in creatine peaks in the prefrontal cortex and cerebellum.In similarity, previous studies have found reduced creatine in the anterior cingulate cortex in schizophrenia [23].
Concerning choline peak, it was significantly increased in visual cortex and dorsolateral prefrontal cortex in new cases of schizophrenia.This was also found by Plitman et al. [24].
Choline-containing compounds act as markers of membrane metabolism [24,25].Choline is present in higher concentrations within glial cells than in neurons and has been investigated in patients with schizophrenia.Elevated levels of these neuro-metabolites have been proposed to reflect glial activation and have been observed in several neuroinflammatory disorders [25].

Limitations of the study
The present study was limited by the relative small sample size, a large number of uncooperative patients they had been excluded from our study, non-long term followup of the patients after medications and assessment of the potential changes in MRS, ASL and their relation to the patients' clinical condition this will open new perspective about the role of advanced MRI techniques in diagnosis and follow-up of mental disorders, and in particular, schizophrenia.

Conclusions
Combination of both ASL and MR spectroscopy can provide better diagnosis and differentiation between cases either it is acute or chronic.
As early detection of schizophrenia is an important tool to improve the outcome of the disease and adjust the

Recommendations
Finally, from the current study, we recommend ASL and MR spectroscopy techniques in screening for psychiatric disease among the high-risk individuals, especially those with positive family history, which will help in early detection of schizophrenic patients and their first-degree relatives even with no obvious clinical symptomatology.
Continued data collection in a larger sample, longer duration with the use of powerful MRI 3.0-Tesla scanners will be helpful in further studies to develop the best practice for ASL and MR spectroscopy.
Follow-up studies for longer period up to 6-12 months will be valuable in treatment assessment and remodelling treatment plan according to course of the disease for every patient.

Fig. 1
Fig. 1 ROC analysis for the diagnostic performance of ASL, A ROC curve for ASL performance at the anterior cingulate cortex, B ROC curve for ASL performance at the parietal lobe

Fig. 2 A
Fig. 2 A twenty-one-year-old male patient complained of visual Hallucinations and Delusions, on clinical psychiatric examination: correct response on Wisconsin Card Sorting Test (WCST) was 28, errors response on Wisconsin Card Sorting Test (WCST) was 30 and that on Stroop test was 470.A Conventional MRI (A): axial FLAIR MRI brain image shows no significant abnormalities B-ASL (B): Axial ASL colour map shows mildly reduced frontal blood flow, (rCBF = 39.38 ml/min/100g) C-ASL (C) Axial ASL colour map shows mildly reduced parietal lobe blood flow, (rCBF = 39.61ml/min/100g) D-ASL (D) Axial ASL colour map shows mildly increased putamen blood flow, (rCBF = 63.03ml/min/100g)E-ASL (E) Axial ASL colour map shows moderately increased hippocampus blood flow, (rCBF = 70.55ml/min/100g) F-MRS (F) MRS (long TE144) of Anterior Cingulate Cortex shows decreased NAA and reduced glutamate and increased glutamine, with NAA/Choline ratio 0.768 and NAA/Creatine ratio 1.19 G-MRS (G) MRS (long TE144) of Thalamus shows reduced NAA and increased both glutamate and glutamine with NAA/choline ratio 0.71 and NAA/creatine ratio 1.11.Final Radiological diagnosis: a case of acute schizophrenia, matching with clinical tests (See figure on next page.)

(
See figure on next page.)Fig.3A twenty-two-year-old male patient complained of Obsessions, Paranoia and hallucinations (visual and auditory), on clinical psychiatric examination: correct response on Wisconsin Card Sorting Test (WCST) was 27, errors response on Wisconsin Card Sorting Test (WCST) was 33 and that on Stroop test was 440.A Conventional MRI (A): axial FLAIR MRI brain image shows no significant abnormalities B -ASL (B): Axial ASL colour map shows mildly reduced frontal blood flow, (rCBF= 38.36ml/min/100g) C -ASL (C) Axial ASL colour map shows mildly reduced parietal lobe blood flow, (rCBF=39.66ml/min/100g)D -ASL (D) Axial ASL colour map shows moderately increased putamen blood flow, (rCBF = 62.55ml/min/100g ) E -ASL (E) Axial ASL colour map shows moderately increased hippocampus blood flow, (rCBF=66.17ml/min/100g) F-MRS (F) MRS (long TE144) of Frontal lobe shows decreased NAA and reduced glutamate and increased glutamine, with NAA/Choline ratio 1.29 and NAA/Creatine ratio 2.32 G -MRS (G) MRS (long TE144) of Thalamus shows reduced NAA and increased both glutamate and glutamine with NAA/choline ratio 0.92 and NAA/ creatine ratio1.21Final Radiological diagnosis: a case of acute schizophrenia, matching with clinical tests

Table 1
Sociodemographic data of the studied group

Table 2
ASL diagnosis of the studied participants F, p: F and p values for ANOVA test, Sig.bet.Groups were done using Post Hoc Test (LSD) p1: p value for comparing between F.E and Chronic Schizophrenia patients p2: p value for comparing between F.E Schizophrenia patients and Control group p3: p value for comparing between Chronic Schizophrenia patients and Group Control group

Table 3
MRS finding regards to NAA/CR Ratios

Table 5
MRS finding regards to choline/creatine ratio

Table 6
The differences of paired sample area under the ROC curve for the diagnostic performance of ASL AUC: Area Under a Curve, p value: Probability value, CI: Confidence Intervals, NPV: Negative predictive value, PPV: Positive predictive value *Statistically significant at p ≤ 0.05 #Cut off was choose according to Youden index

Table 7
Inter-observer agreement of rCBF values between observers in different brain regions