the study was approved by the ethics committee of the medical university of vienna and was conducted as a pilot study . written informed consent was obtained from all patients . seven patients ( 2 females ; median age at time of first pet scan : 50 years , range 3354 ) with drug - resistant tle were included in this study after surgical treatment with either selective amygdalohippocampectomy ( n = 4 ) or anteromedial temporal lobectomy ( n = 3 ) . patients were recruited from a group of 9 patients studied preoperatively with vpm - pet ; 2 previous patients were not included because one patient was not available for follow - up after surgery and the other had no arterial input function for presurgery pet . a second vpm - pet scan was performed a median of 42 months ( range 1754 ) after surgery ( see table for details ) . surgery outcome in individual patients was ranked according to ( 1 ) complete seizure freedom after surgery , ( 2 ) type and number of seizures during follow - up period after surgery , and ( 3 ) intake of aeds ( figure e-1 on the neurology web site at neurology.org ) . clinical , eeg , and mri data for patients with drug - resistant tle who underwent surgery dynamic vpm - pet scans were acquired on an advance pet scanner ( ge medical systems , waukesha , wi ) as described before , with arterial blood sampled throughout . radiolabeled metabolites of vpm were measured in discrete arterial blood samples using a previously described solid - phase extraction assay . for all patients , t1-weighted mris were acquired before and after surgery with an achieva 3.0 t scanner ( philips medical systems , best , the netherlands ) . six tl volumes of interest ( vois ) ( amygdala , parahippocampal gyrus , anterior temporal lobe , middle and inferior temporal gyrus , superior temporal gyrus , and posterior temporal lobe ) and 2 extratemporal vois ( superior parietal gyrus and cerebellum ) were chosen for analysis . the hippocampus itself could not be analyzed because of spillover of radioactivity from the adjacent choroid plexus , which showed high vpm uptake . kinetic modeling of pet data was performed by using an arterial input function corrected for polar radiolabeled metabolites of vpm and a 1-tissue 2rate constant compartment model to derive the transfer rate constant k1 ( ml / min / cm ) of vpm from plasma into brain as an outcome parameter of pgp function , with low k1 indicating high pgp function . to minimize the influence of radiolabeled metabolites of vpm on k1 estimates , only the first 10 minutes of the pet data brain tissue ( hippocampus for all patients , and anterior - mesial parts of the tl of 3 patients ) was removed during epilepsy surgery and fixed in 10% neutral buffered formalin immediately after surgery . for immunohistochemical staining , brain sections were prepared and probed with antibodies recognizing pgp ( clones , jsb1 1:400 , c219 1:80 , c494 1:1,000 ; alexis biochemicals , lausen , switzerland ) followed by quantitative image assessment as published . statistical testing was performed using prism 5.0 software ( graphpad software , la jolla , ca ) . data are presented as mean  sd for pet data and median ( range ) for demographic data . friedman test with dunn post hoc test was used when multiple groups were compared , and wilcoxon matched - pairs signed rank test was used when 2 groups were compared . the study was approved by the ethics committee of the medical university of vienna and was conducted as a pilot study . written informed consent was obtained from all patients . seven patients ( 2 females ; median age at time of first pet scan : 50 years , range 3354 ) with drug - resistant tle were included in this study after surgical treatment with either selective amygdalohippocampectomy ( n = 4 ) or anteromedial temporal lobectomy ( n = 3 ) . patients were recruited from a group of 9 patients studied preoperatively with vpm - pet ; 2 previous patients were not included because one patient was not available for follow - up after surgery and the other had no arterial input function for presurgery pet . a second vpm - pet scan was performed a median of 42 months ( range 1754 ) after surgery ( see table for details ) . surgery outcome in individual patients was ranked according to ( 1 ) complete seizure freedom after surgery , ( 2 ) type and number of seizures during follow - up period after surgery , and ( 3 ) intake of aeds ( figure e-1 on the neurology web site at neurology.org ) . clinical , eeg , and mri data for patients with drug - resistant tle who underwent surgery dynamic vpm - pet scans were acquired on an advance pet scanner ( ge medical systems , waukesha , wi ) as described before , with arterial blood sampled throughout . radiolabeled metabolites of vpm were measured in discrete arterial blood samples using a previously described solid - phase extraction assay . for all patients , t1-weighted mris were acquired before and after surgery with an achieva 3.0 t scanner ( philips medical systems , best , the netherlands ) . six tl volumes of interest ( vois ) ( amygdala , parahippocampal gyrus , anterior temporal lobe , middle and inferior temporal gyrus , superior temporal gyrus , and posterior temporal lobe ) and 2 extratemporal vois ( superior parietal gyrus and cerebellum ) were chosen for analysis . the hippocampus itself could not be analyzed because of spillover of radioactivity from the adjacent choroid plexus , which showed high vpm uptake . kinetic modeling of pet data was performed by using an arterial input function corrected for polar radiolabeled metabolites of vpm and a 1-tissue 2rate constant compartment model to derive the transfer rate constant k1 ( ml / min / cm ) of vpm from plasma into brain as an outcome parameter of pgp function , with low k1 indicating high pgp function . to minimize the influence of radiolabeled metabolites of vpm on k1 estimates , only the first 10 minutes of the pet data brain tissue ( hippocampus for all patients , and anterior - mesial parts of the tl of 3 patients ) was removed during epilepsy surgery and fixed in 10% neutral buffered formalin immediately after surgery . for immunohistochemical staining , brain sections were prepared and probed with antibodies recognizing pgp ( clones , jsb1 1:400 , c219 1:80 , c494 1:1,000 ; alexis biochemicals , lausen , switzerland ) followed by quantitative image assessment as published . statistical testing was performed using prism 5.0 software ( graphpad software , la jolla , ca ) . data are presented as mean  sd for pet data and median ( range ) for demographic data . friedman test with dunn post hoc test was used when multiple groups were compared , and wilcoxon matched - pairs signed rank test was used when 2 groups were compared . in all patients , vpm - k1 values from preoperative vpm - pet scans were lower in regions pathophysiologically connected to the epileptic focus ( amygdala : 0.032  0.011 ; superior temporal gyrus : 0.037  0.012 ) compared with a reference region considered not to be involved in epileptogenic pathways ( cerebellum : 0.045  0.012 ) ( figure 1a ) . moreover , vpm - k1 was lower in amygdala than in posterior tl ( 0.043  0.011 ) . there were no differences between tl vpm - k1 values ipsilaterally or contralaterally to the seizure focus . ( a ) vpm - k1 values in different brain regions located ipsilateral to the seizure focus of individual patients before epilepsy surgery ( * * * p < 0.001 , * * p < ( c ) correlation of percentage area of pgp immunopositive labeling in the surgically resected hippocampus with vpm - k1 values in ipsilateral anterior tl ( rs = spearman rank correlation coefficient ) . ( d ) vpm - k1 values in different brain regions located ipsilateral to the seizure focus of individual patients after epilepsy surgery . brain regions affected by surgery are not shown ( * p < 0.05 ; friedman test with dunn post hoc test ) . amyg = amygdala ; ant - tl = anterior temporal lobe ; mitg = middle and inferior temporal gyrus ; p = patient ; para - hc = parahippocampal gyrus ; pgp = p - glycoprotein ; post - tl = posterior temporal lobe ; spg = superior parietal gyrus ; stg = superior temporal gyrus ; tl = temporal lobe ; vpm = ( r)-[c]verapamil . pgp immunoreactivity in surgically resected hippocampal specimens was determined using pgp - specific antibodies ( figure 1b ) . the higher the pgp immunoreactivity in surgically resected hippocampal specimens , the lower were the ipsilateral vpm - k1 values for anterior tl ( rs = 0.821 , p = 0.034 ; figure 1c ) and superior temporal gyrus ( rs = 0.857 , p = 0.024 ) . postoperative vpm - k1 values were lower in superior temporal gyrus ( 0.033  0.007 ) compared with cerebellum ( 0.041  0.008 ) and the posterior tl ( 0.041  0.009 ; figure 1d ) . the aed load was reduced in all patients , with 2 patients coming off all aeds , but this did not affect peripheral metabolism of vpm ; i.e. , fractions of polar radiolabeled metabolites of vpm in plasma at 10 minutes after injection were similar before ( 0.140  0.039 ) and after surgery ( 0.136  0.042 , p = 0.938 ) . patients with better outcome had lower mean presurgical vpm - k1 values in ipsilateral tl ( higher pgp function ) than patients with poorer outcome ( rs = 0.837 , p = 0.024 ) ( figure 2a ) . patients with better outcome had high areas of pgp immunopositive labeling in surgically resected hippocampal specimens , while patients with poorer outcome had low pgp immunoreactivity ( rs = 0.782 , p = 0.048 ; figure 2b ) . in the 3 patients with best postoperative outcome ( patients 1 and 6 : seizure - free and without medication ; patient 3 : seizure - free and with very low aed load ) , vpm - k1 values increased relative to preoperative pet scans in all brain vois studied , whereas k1 values decreased in patients with worst outcome ( figure 2c ) . ( a ) correlation of mean presurgical vpm - k1 values in ipsilateral tl with surgery outcome rank in individual patients ( see the methods section and figure e-1 for definition of surgery outcome rank order ; rs = spearman rank correlation coefficient ) . ( b ) correlation of percentage area of pgp immunopositive labeling in the surgically resected hippocampus with surgery outcome rank in individual patients . ( c ) mean percent change in vpm - k1 values across all studied tl regions located ipsilateral to the seizure focus in postsurgery pet scan relative to presurgery pet scan . patients 3 and 4 were both assigned rank 3 , but the antiepileptic drug load of patient 3 was lower than that of patient 4 . p = patient ; pgp = p - glycoprotein ; tl = temporal lobe ; vpm = ( r)-[c]verapamil . in all patients , vpm - k1 values from preoperative vpm - pet scans were lower in regions pathophysiologically connected to the epileptic focus ( amygdala : 0.032  0.011 ; superior temporal gyrus : 0.037  0.012 ) compared with a reference region considered not to be involved in epileptogenic pathways ( cerebellum : 0.045  0.012 ) ( figure 1a ) . moreover , vpm - k1 was lower in amygdala than in posterior tl ( 0.043  0.011 ) . there were no differences between tl vpm - k1 values ipsilaterally or contralaterally to the seizure focus . ( a ) vpm - k1 values in different brain regions located ipsilateral to the seizure focus of individual patients before epilepsy surgery ( * * * p < 0.001 , * * p < ( c ) correlation of percentage area of pgp immunopositive labeling in the surgically resected hippocampus with vpm - k1 values in ipsilateral anterior tl ( rs = spearman rank correlation coefficient ) . ( d ) vpm - k1 values in different brain regions located ipsilateral to the seizure focus of individual patients after epilepsy surgery . brain regions affected by surgery are not shown ( * p < 0.05 ; friedman test with dunn post hoc test ) . amyg = amygdala ; ant - tl = anterior temporal lobe ; mitg = middle and inferior temporal gyrus ; p = patient ; para - hc = parahippocampal gyrus ; pgp = p - glycoprotein ; post - tl = posterior temporal lobe ; spg = superior parietal gyrus ; stg = superior temporal gyrus ; tl = temporal lobe ; vpm = ( r)-[c]verapamil . pgp immunoreactivity in surgically resected hippocampal specimens was determined using pgp - specific antibodies ( figure 1b ) . the higher the pgp immunoreactivity in surgically resected hippocampal specimens , the lower were the ipsilateral vpm - k1 values for anterior tl ( rs = 0.821 , p = 0.034 ; figure 1c ) and superior temporal gyrus ( rs = 0.857 , p = 0.024 ) . postoperative vpm - k1 values were lower in superior temporal gyrus ( 0.033  0.007 ) compared with cerebellum ( 0.041  0.008 ) and the posterior tl ( 0.041  0.009 ; figure 1d ) . the aed load was reduced in all patients , with 2 patients coming off all aeds , but this did not affect peripheral metabolism of vpm ; i.e. , fractions of polar radiolabeled metabolites of vpm in plasma at 10 minutes after injection were similar before ( 0.140  0.039 ) and after surgery ( 0.136  0.042 , p = 0.938 ) . patients with better outcome had lower mean presurgical vpm - k1 values in ipsilateral tl ( higher pgp function ) than patients with poorer outcome ( rs = 0.837 , p = 0.024 ) ( figure 2a ) . patients with better outcome had high areas of pgp immunopositive labeling in surgically resected hippocampal specimens , while patients with poorer outcome had low pgp immunoreactivity ( rs = 0.782 , p = 0.048 ; figure 2b ) . in the 3 patients with best postoperative outcome ( patients 1 and 6 : seizure - free and without medication ; patient 3 : seizure - free and with very low aed load ) , vpm - k1 values increased relative to preoperative pet scans in all brain vois studied , whereas k1 values decreased in patients with worst outcome ( figure 2c ) . ( a ) correlation of mean presurgical vpm - k1 values in ipsilateral tl with surgery outcome rank in individual patients ( see the methods section and figure e-1 for definition of surgery outcome rank order ; rs = spearman rank correlation coefficient ) . ( b ) correlation of percentage area of pgp immunopositive labeling in the surgically resected hippocampus with surgery outcome rank in individual patients . ( c ) mean percent change in vpm - k1 values across all studied tl regions located ipsilateral to the seizure focus in postsurgery pet scan relative to presurgery pet scan . patients 3 and 4 were both assigned rank 3 , but the antiepileptic drug load of patient 3 was lower than that of patient 4 . p = patient ; pgp = p - glycoprotein ; tl = temporal lobe ; vpm = ( r)-[c]verapamil . in a longitudinal pilot vpm - pet study in patients with drug - resistant tle before and after tl surgery with a median follow - up period after surgery of 6 years ( range 47 ) , we detected a our preoperative in vivo pet findings are supported by direct correlation with pgp immunoreactivity in surgical specimens in the same patients . furthermore , high pgp activity in epileptogenic pathways preoperatively was associated with better postoperative outcome . our longitudinal pet data suggest that pgp overactivity is dynamic rather than static , and seizure freedom and reduction in aed load are associated with reversal of upregulation . in line with the transporter hypothesis of drug resistance , we found significantly lower vpm - k1 values in some tl brain regions close to the presumed epileptic focus ( amygdala , superior temporal gyrus ) as compared with a reference region outside the tl ( cerebellum ) or with a region more distant from the focus ( posterior tl ) , pointing to pgp overactivity in epileptogenic brain tissue . consistent with previous findings , we show that pgp overactivity was not restricted to the ipsilateral tl , but also extended to the contralateral side involved in epileptogenic pathways . after epilepsy surgery , patients with optimal surgery outcome defined as seizure freedom and withdrawal of aeds had global vpm - k1 increases relative to presurgery pet scans , suggestive of decreased pgp function . this  return to normal  after successful epilepsy surgery is consistent with our recent report of higher vpm - k1 values in pharmacosensitive compared with drug - refractory patients and with findings in postmortem brain tissue , which showed almost no pgp overexpression in the sclerotic hippocampus of patients with epilepsy who had entered terminal remission before death . a previous immunohistochemistry study showed higher pgp expression in the tl white matter of patients with postoperative seizure relapse compared with postoperatively seizure - free patients , in keeping with our finding of increased pgp activity postoperatively in patients with poorer outcome . our preoperative in vivo pet data and ex vivo immunohistochemistry , however , suggest that pgp overactivity in epileptogenic pathways and hippocampal overexpression are associated with optimal outcome after tl surgery . this discrepancy in predicting postoperative outcome might be explained by methodologic differences : we quantified hippocampal pgp immunoreactivity , whereas kwan et al . used semiquantitative approaches and found differences in pgp immunoreactivity in tl white matter , but also a trend toward higher neuronal pgp expression in postoperatively seizure - free patients . the latter finding is in keeping with our in vivo pet finding of pgp overactivity in tl gray matter as a predictor of optimal outcome . our pilot study was not designed to assess the predictive ability of vpm - pet as biomarker of surgery outcome ; this should be tested prospectively in a larger cohort , and integrated with other known risk factors for less favorable outcome , such as psychiatric comorbidities and incompleteness of resection . moreover , our study can not distinguish between the effects of seizure cessation from dose decrease or discontinuation of aeds on postoperative pgp function . seizure - free ( n = 4 ) vs not seizure - free ( n = 3 ) patients , or seizure - free patients without aeds ( n = 2 ) vs seizure - free patients on aeds ( n = 2 ) , with meaningful results . alternatively , one could conduct a prospective study to leave the patients on their medication for the first year after surgery and to acquire 2 separate postsurgery pet scans , before and after the change in medication . however , our data suggest that aed exposure also affects pgp activity , because our outcome ranking mainly based on seizure burden after surgery correlates with aed load . another limitation of this study is that we were not able to obtain vpm - k1 values in hippocampus , which would require the availability of a pet scanner with higher spatial resolution and advanced image reconstruction to overcome the spillover from the high vpm uptake in the adjacent choroid plexus . our study can not address whether pgp overactivity is the cause of drug resistance in tle , and thus  surgical  removal of this overactivity contributed to seizure freedom , or whether upregulation is a marker of seizure burden rather than a cause . in both scenarios , pgp overactivity may reduce target concentrations of aeds and thereby possibly contribute to drug resistance . our study demonstrates that pgp overactivity is not static , and thus could be therapeutically targeted by drugs that inhibit or downregulate pgp to possibly prevent development of , or bring about reversal of , drug resistance mediated by pgp . our data confirm previous findings that vpm - pet reflects pgp function in vivo in patients with tle , and that cerebral pgp overactivity is associated with drug resistance in tle . our postoperative vpm - pet data extend previous findings , indicating a  normalization  of pgp function in those patients who benefited most from surgery . our findings also suggest that pgp overactivity in the tl on preoperative vpm - pet might be indicative of optimal postoperative outcome . dr . bauer : study concept or design , acquisition of data , analysis or interpretation of data , drafting / revising the manuscript for content . karch : analysis or interpretation of data , statistical analysis , drafting / revising the manuscript for content . liu : acquisition of data , analysis or interpretation of data , drafting / revising the manuscript for content . hainfellner : analysis or interpretation of data , drafting / revising the manuscript for content . pataraia : study concept or design , analysis or interpretation of data , drafting / revising the manuscript for content . langer : study concept or design , acquisition of data , analysis or interpretation of data , drafting / revising the manuscript for content . supported by the european community 's seventh framework program ( grant 201380 , to m.j.k , s.m.s . , m .- c.a . , m. mller , and o.l . ) , the austrian science fund ( fwf ) ( grant f 3513-b20 , to m. mller and o.l . , and kli 139-b00 , to m. mller and o.l . ) , and the department of health 's national institute for health research biomedical research centres funding scheme ( to m.j.k . and s.m.s . ) . m. bauer , r. karch , m. zeitlinger , and j. liu report no disclosures relevant to the manuscript . m. koepp is funded by the european community 's seventh framework program ( grant 201380 , expired ) . dr koepp served on a scientific advisory board of ge healthcare , and has received honoraria for lectures from ucb pharma , eisai , novartis , and desitin . m. asselin is funded by the european community 's seventh framework program ( grant 201380 , expired ) . s. sisodiya is funded by the european community 's seventh framework program ( grant 201380 , expired ) . dr . sisodiya served on a scientific advisory board for ucb pharma , and has received institutional support or honoraria for lectures from ucb pharma , gsk , and eisai . j. hainfellner , w. wadsak , and m. mitterhauser report no disclosures relevant to the manuscript . m. mller is funded by the european community 's seventh framework program ( grant 201380 , expired ) and the austrian science fund ( fwf ) ( grants f 3513-b20 and kli 139-b00 ) . o. langer is funded by the european community 's seventh framework program ( grant 201380 , expired ) and the austrian science fund ( fwf ) ( grants f 3513-b20 and kli 139-b00 ) .
objectives : to study the functional activity of the multidrug efflux transporter p - glycoprotein ( pgp ) at the blood - brain barrier of patients with temporal lobe epilepsy using ( r)-[11c]verapamil ( vpm)-pet before and after temporal lobe surgery to assess whether postoperative changes in seizure frequency and antiepileptic drug load are associated with changes in pgp function.methods:seven patients with drug - resistant temporal lobe epilepsy underwent vpm - pet scans pre- and postsurgery . 
 patients were followed up for a median of 6 years ( range 47 ) after surgery . 
 pgp immunoreactivity in surgically resected hippocampal specimens was determined with immunohistochemistry.results:optimal surgical outcome , defined as seizure freedom and withdrawal of antiepileptic drugs , was associated with higher temporal lobe pgp function before surgery , higher pgp - positive staining in surgically resected hippocampal specimens , and reduction in global pgp function postoperatively , compared with nonoptimal surgery outcome.conclusions:the data from our pilot study suggest that pgp overactivity in epilepsy is dynamic , and complete seizure control and elimination of antiepileptic medication is associated with reversal of overactivity , although these findings will require confirmation in a larger patient cohort .