Data from 36 patients in the TRS group before commencing clozapine (Table 1: 27 [75%] males, 9 [15%] females; 30 schizophrenia, 6 schizoaffective disorder; mean age 39.47 years [SD 13.82; range 19-68]) and 16 healthy volunteers (10 [62.5%] males, 6 [37.5%] females; mean age 30.69 years [SD 10.77; range 21-54]) were available for analyses. The healthy volunteers were younger than the TRS group (t(50)=2.252, p=0.029), while sex did not differ by group (p>0.05). Among the TRS group, complete data (ASL, structural images and PANSS scores at baseline and 12 weeks) were available in 24 individuals (Table 3). Symptom severity scores for all (sub)scales were significantly lower after 12 weeks of clozapine treatment compared to the baseline values (Table 3).
Table 1. Descriptive statistics of the patient sample at baseline and follow-up
|
Baseline
|
Week 12 Follow-up
|
|
N=36
|
N=24
|
Age, years
|
39.47 (13.82)
|
38.83 (13.04)
|
Sex, Male/Female
|
27/9
|
18/6
|
Age of onset, years
|
26.11 (9.00)
|
26.13 (8.68)
|
Duration of illness, years
|
14.31 (8.77)
|
13.92 (8.99)
|
Previous clozapine use, Yes/No
|
7/29
|
4/20
|
Diagnosis, F20-schizophrenia /F25-schizoaffective
|
30/6
|
21/3
|
|
N=35
|
|
Previous antipsychotic trials,
min; max; median; range
|
2; 10; 3; 8
|
2; 7; 3; 5
|
Antipsychotic dose,
CPZE/mg per day
|
229.80 (184.70)
|
/
|
Number of hospital admissions, min; max; median; range
|
0; 12; 3; 12
|
0; 12; 3; 12
|
Symptoms and Functioning
|
N=36
|
|
PANSS-Positive
|
18.43 (5.87)
|
14.33 (5.10)
|
PANSS-Negative
|
19.23 (7.51)
|
15.29 (5.57)
|
PANSS-General
|
35.33 (6.94)
|
27.04 (5.98)
|
PANSS-Total
|
72.97 (14.62)
|
56.29 (14.11)
|
GAF
|
45.29 (10.51)
|
59.42 (8.66)
|
Clozapine
|
|
N=21
|
Clozapine dose, daily mg
|
/
|
370.2 (144.18)
|
Plasma clozapine, ng/ml
|
/
|
0.48 (0.32)
|
Plasma norclozapine, ng/ml
|
/
|
0.24 (0.13)
|
Data shown as Mean (Standard Deviation) unless otherwise specified. CPZE, Chlorpromazine equivalent dose; GAF, Global Assessment of Functioning; PANSS, Positive and Negative Syndrome Scale.
CBF in TRS compared to healthy volunteers.
ROI analysis
There was a significant main effect for Group (F(1,50)=7.488, p=0.009), related to lower CBF in the TRS group than in the healthy volunteer group in all ROIs (Table 2; Figure 1). The Group * ROI interaction was non-significant (p>0.05). However, global CBF was also lower in the TRS group (t(50)=-2.73, p=0.01). When age and global CBF were included as covariates in the model, the main effect of ‘Group’ was no longer significant (F(1,48)=0.323, p>0.05).
Whole-brain voxel-wise analysis
After controlling for age and global CBF, voxel-wise comparison revealed significantly lower CBF in the TRS group than in healthy volunteers in several areas (Table S1; Figure 1), including the frontal (four clusters: MNI (x, y, z) =32, 22, 2, p=0.012 FWE; MNI (x, y, z) =-8, 38, 30, p=0.001 FWE; MNI (x, y, z) =-46, 12, 40, p=0.034 FWE; MNI (x, y, z) =-20, 22, 58, p=0.021 FWE), temporal (one cluster: MNI (x, y, z) =-26, -42, 2, p=0.010 FWE), and parietal (one cluster: MNI (x, y, z) =40, -62, 48, p=0.000 FWE) cortices. The opposite contrast (i.e., TRS group > Healthy Volunteer group) revealed no significant clusters.
Table 2. Analysis of Cerebral Blood Flow in Treatment Resistant Schizophrenia Prior to Clozapine Initiation and Healthy Volunteers in Selected ROIs
|
TRS (N=36)
|
HV (N=16)
|
No Covariates
|
|
|
|
Test statistics
|
%
|
d
|
|
|
|
|
|
|
|
|
Global CBF
|
38.83 (10.45)
|
48.08 (12.99)
|
-2.73
|
50
|
0.01
|
21.29%
|
-0.82
|
Regional CBF
|
|
|
|
|
|
|
|
ACC
|
37.10 (10.96)
|
46.88 (13.56)
|
7.49
|
1
|
0.01
|
23.29%
|
-0.83
|
Striatum
|
40.83 (7.64)
|
47.36 (9.16)
|
14.81%
|
-0.80
|
Thalamus
|
39.04 (8.08)
|
45.91 (8.94)
|
16.17%
|
-0.82
|
Hipp
|
41.61 (8.08)
|
47.39 (10.15)
|
12.99%
|
-0.66
|
Data shown as Mean (Standard Deviation). ACC, Anterior Cingulate Cortex; CBF, Cerebral Blood Flow; Hipp, Hippocampus; HV, Healthy Volunteer; TRS, Treatment Resistant Schizophrenia; %, Percentage Difference between Groups.
Changes in CBF during clozapine treatment
ROI analysis
Analysis of change in CBF during clozapine treatment found a significant main effect of ROI (F(3,69)=21.126, p<0.001) and significant Time * ROI interaction (F(3,69)=5.059, p=0.003). These effects remained significant when covarying for change in global CBF (main effect of ROI: F(3,66)=21.956, p<0.001; Time * ROI interaction: F(3,66)=4.388, p=0.007). Subsequent post hoc tests in each ROI separately found regional CBF was significantly decreased in the ACC after 12 weeks of clozapine treatment compared to baseline (t(23)=2.943, p=0.007; Figure 2) which remained significant when covarying for the numerical differences in global CBF over time (F(1,22)=11.177, p=0.003; Table 3). There were no significant changes in regional CBF in the hippocampus, striatum, and thalamus or globally during clozapine treatment (Table 3).
Whole-brain voxel-wise analysis
Whole-brain voxel-wise analysis (Table S2) also revealed significantly decreased regional CBF in the ACC in the TRS group after 12 weeks of clozapine treatment (one cluster: MNI (x, y, z) = -2, 54, 30, p=0.006 FWE; Figure 2). This effect remained significant when controlling for changes in global CBF (one cluster: MNI (x, y, z) =-2, 52, 30, p=0.001 FWE; Figure 2). The opposite contrast (i.e., Baseline < Week 12 Follow-up) did not reveal any significant clusters without covarying with changes in global CBF. After controlling for changes in global CBF this contrast revealed significantly increased CBF following clozapine treatment compared to baseline in the left postcentral area (one cluster: MNI (x, y, z) =-58, -6, 22, p=0.017 FWE; Figure 2).
Table 3. Test Statistics of the patient sample at baseline and follow-up
|
Patient Sample (N=24)
|
No Covariates
|
Covarying for percent change of Global CBF
|
|
Baseline
|
Week 12
|
|
|
Test statistics
|
Test statistics
|
|
|
|
%
|
d
|
|
|
|
|
|
|
Global CBF
|
38.62 (10.48)
|
36.26 (8.64)
|
6.30%
|
0.35
|
3.00
|
1
|
0.10
|
/
|
/
|
/
|
Regional CBF
|
|
|
|
ACC
|
37.12 (10.72)
|
33.37 (8.35)
|
10.64%
|
0.60
|
8.66
|
1
|
0.01
|
11.18
|
1
|
0.00
|
Striatum
|
40.43 (7.37)
|
39.44 (7.49)
|
2.48%
|
0.18
|
0.76
|
1
|
0.39
|
0.07
|
1
|
0.80
|
Thalamus
|
38.55 (8.68)
|
36.87 (8.31)
|
4.46%
|
0.26
|
1.61
|
1
|
0.22
|
0.04
|
1
|
0.84
|
Hipp
|
42.01 (8.01)
|
41.34 (7.98)
|
1.61%
|
0.10
|
0.25
|
1
|
0.62
|
1.44
|
1
|
0.24
|
Symptom Severity and Functioning
|
PANSS-Positive
|
18.92 (5.98)
|
14.33 (5.10)
|
27.61%
|
1.75
|
73.47
|
1
|
<0.001
|
/
|
/
|
/
|
PANSS-Negative
|
18.13 (6.42)
|
15.29 (5.57)
|
17.00%
|
0.61
|
8.84
|
1
|
0.01
|
/
|
/
|
/
|
PANSS-General
|
34.54 (7.44)
|
27.04 (5.98)
|
24.36%
|
1.26
|
38.15
|
1
|
<0.001
|
/
|
/
|
/
|
PANSS-Total
|
71.58 (15.99)
|
56.29 (14.11)
|
23.91%
|
1.56
|
58.54
|
1
|
<0.001
|
/
|
/
|
/
|
GAF
|
48.25 (9.31)
|
59.42 (8.66)
|
20.75%
|
-1.10
|
29.00
|
1
|
<0.001
|
/
|
/
|
/
|
Data shown as Mean (Standard Deviation). ACC, Anterior Cingulate Cortex; CBF, Cerebral Blood Flow; GAF, Global Assessment of Functioning; Hipp, Hippocampus; PANSS, Positive and Negative Syndrome Scale; %, Percentage Difference between Groups.
Relationships between change in CBF and symptom improvement
The change in regional CBF in the ACC and thalamus was significantly associated with the percentage change in symptom scores over 12 weeks of clozapine treatment (Table S3), such that greater reductions in ACC CBF were associated with lesser improvement in negative symptom scores (r=-0.493, df=21, p=0.017, covarying for change in global CBF; Figure S1) and greater reductions in thalamus CBF were associated with lesser improvement in positive (r=-0.417, df=21, p=0.048, covarying for change in global CBF; Figure S21) and total (r=-0.494, df=21, p=0.017, covarying for change in global CBF; Figure S22) symptom scores. There were no significant relationships between plasma clozapine level or clozapine dose at 12 weeks and the percent change in CBF (all p>0.05). Voxel-wise analyses did not detect any significant associations between the change in CBF and symptom scores over 12 weeks, plasma clozapine levels or clozapine dose.
Relationship between CBF at baseline and symptom improvement
Baseline regional CBF in the striatum and hippocampus were significantly associated with subsequent percentage change in symptom scores (Table S4), such that participants with higher baseline striatum CBF had greater improvements in general symptom scores (r=0.403, df=25, p=0.037, covarying for baseline global CBF; Figure S3), and participants with higher baseline hippocampus CBF had greater improvements in total (r=0.496, df=25, p=0.009, covarying for baseline global CBF; Figure S41) and positive (r=0.403, df=25, p=0.037, covarying for baseline global CBF; Figure S42) symptom scores. There were no significant relationships between CPZE level and ROI CBF at baseline (all p>0.05). No further significant correlations between ROI CBF and clinical variables were detected, and voxel-wise analyses did not find significant associations between clinical variables and baseline CBF.
GM volume
GM volume in TRS compared to in healthy volunteers.
Whole brain GM volume (Table S5) in the TRS group was significantly lower than in the healthy volunteer group (t(50)=-0.792, p=0.007; Figure S5). After controlling for age, sex and TIV, this effect remained significant (F(1)=8.881, p=0.005). Voxel-wise comparison revealed significantly lower global GM volume in the TRS group than in healthy volunteers in several regions including the ACC, thalamus, and hippocampus (Table S1, Figure S5). The opposite contrast (i.e., TRS group > Healthy Volunteer group) revealed no significant clusters.
Changes in GM volume during clozapine treatment
Within the TRS group, whole brain GM volume (Table S6) was significantly decreased after 12 weeks of clozapine treatment compared to baseline (t(23)=4.043, p<0.001; Figure S6). Whole-brain voxel-wise analysis also revealed decreases in GM volume, including in the frontal, temporal, occipital and parietal cortices, caudate, putamen and cerebellum (Table S2; Figure S6). The opposite contrast (i.e., Baseline < Week 12 Follow-up) revealed no significant clusters.
Effect of GM volume on CBF
Reductions in CBF in TRS compared to healthy volunteers remained similar after controlling for GM volume (Table S1, Figure S7). Reductions in CBF during clozapine treatment in frontal regions also remained significant after controlling for change in grey matter volume, but the increase in CBF in the left postcentral area was no longer significant (Table S2, Figure S7).