Patients with neurologic complaints are imaged with MRI protocols that may include many pulse sequences. It has not been documented which sequences are essential. We assessed the diagnostic accuracy of a limited number of sequences in patients with new neurologic complaints.996 consecutive brain MRI studies from patients with new neurological complaints were divided into 2 groups. In group 1, reviewers used a 3-sequence set that included sagittal T1-weighted, axial T2-weighted fluid-attenuated inversion recovery, and axial diffusion-weighted images. Subsequently, another group of studies were reviewed using axial susceptibility-weighted images in addition to the 3 sequences. The reference standard was the study's official report. Discrepancies between the limited sequence review and the reference standard including Level I findings (that may require immediate change in patient management) were identified.There were 84 major findings in 497 studies in group 1 with 21 not identified in the limited sequence evaluations: 12 enhancing lesions and 3 vascular abnormalities identified on MR angiography. The 3-sequence set did not reveal microhemorrhagic foci in 15 of 19 studies. There were 117 major findings in 499 studies in group 2 with 19 not identified on the 4-sequence set: 17 enhancing lesions and 2 vascular lesions identified on angiography. All 87 Level I findings were identified using limited sequence .A 4-pulse sequence brain MRI study is sufficient to evaluate patients with a new neurological complaint except when contrast or angiography is indicated. The volume of brain MR studies performed may continue to rise in the future due to the expansion of health care insurance under the Patient Protection and Affordable Care Act (PPACA) and as a result of an aging population. At our institution, patients with various neurologic complaints are often imaged with MRI protocols that may involve a large number of pulse sequences, resulting in long scan times. There have been major advances in MRI hardware and software technology since the days of the first nuclear magnetic resonance (NMR) images in 1974. These advancements have provided us with an array of available MR sequences to choose from for our neuroimaging protocols. Long scan times related to multiple sequence acquisitions likely result in patient inconvenience and discomfort. It is unclear whether the multiple MR sequences performed are necessary and which of the sequences add value to the interpretation of the study. Optimizing the number of sequences in a routine screening brain MR protocol could improve efficiency as well as increase patient compliance and satisfaction by reducing scan time. Our goal was to investigate the diagnostic accuracy of brain MRI protocols containing a limited number of basic sequences in the evaluation of patients with new neurologic complaints.Diagnostic medical imaging is one of the major driving forces behind rising healthcare expenditures. Between 2000 and 2006, Medicare spending on imaging services more than doubled, increasing to $14 billionThis retrospective study was approved by the Partners Human Research Committee, our hospital's Institutional Review Board (IRB). Consent was not obtained and patient records/information was anonymized and de-identified prior to analysis.We performed a retrospective study of consecutive brain MR examinations performed at our institution, a major urban academic medical center, between February 20, 2010 and April 21, 2010. Studies included patients of all ages who underwent brain MR imaging for the evaluation of new neurologic complaints for which there was no established diagnosis stated on the radiology requisition. We included studies referred from the emergency department, inpatient and outpatient settings. Both intravenous contrast- enhanced and unenhanced studies were included as were studies that contained MR angiogram (MRA) and MR venogram (MRV) sequences. Image review commenced in June 2012, at least 24 months after the examinations were performed. Exclusion criteria were MR studies performed for follow-up evaluation of pre-existing neurological conditions  and specialized MR protocols .All MRI examinations were performed on either 1.5T (Tesla) or 3T MR systems . A total of 9 MRI scanners were used during the period that was reviewed. Eight scanners had field strengths of 1.5T. Six were manufactured by General Electric; 3 were located within the hospital and 3 were located at free standing imaging centers. Two 1.5T MRI scanners were manufactured by Siemens and were located within the hospital. Additionally, a single Siemens 3T scanner was located within the hospital. All MR images in the study were reviewed on a PACS system .The total number of consecutive MRI studies was partitioned into two separate data sets .The group 1 neuroradiologists reviewed a three-sequence abbreviated image set composed of sagittal T1-weighted images, axial T2-weighted FLAIR images, and images derived from a single diffusion-weighted imaging sequence, which included images with and without diffusion gradients, as well as derived apparent diffusion coefficient maps. The neuroradiologists interpreted those three MRI sequences while blinded to both the additional MRI sequences that were acquired, and to the official radiologic report. For each case, a group 1 neuroradiologist formulated an impression based on the three limited sequences and then subsequently compared it to the unblinded impression in the reference official radiologic report. For any discrepancy between the two interpretations, the neuroradiologist was instructed to revisit the limited MR sequences and determine whether, in retrospect, the discrepant findings could have been made on the limited sequences, but were missed due to perceptual error.The second phase of the study involved a different set of staff neuroradiologists with experience ranging from 2 to 20 years. The group 2 neuroradiologists reviewed a four-sequence abbreviated image set composed of the three basic sequences that were reviewed by the group 1 neuroradiologists, with the addition of an axial magnetic susceptibility sensitive sequence , or susceptibility weighted imaging (SWI), including susceptibility weighted angiography (SWAN)). The neuroradiologists interpreted those four MRI sequences while blinded to both the additional MRI sequences that were acquired, and to the official radiologic report. For each case, a group 2 neuroradiologist formulated an impression based on the four limited sequences and then subsequently compared it to the unblinded impression in the official radiologic report, (the reference standard). For any discrepancy between the two interpretations, the neuroradiologist revisited the limited MR sequences and determined whether, in retrospect, the discrepant findings could have been appreciated on the limited sequences, but were missed due to perceptual error.The limited sequence interpretations by the neuroradiologists in groups 1 and 2 were performed on the same PACS workstations as the original interpretations, and they are located in the same interpretation areas as in the original clinical interpretation. The only history that was permitted to the new study reviewer was the limited one provided with the original request for imaging. The neuroradiologists in groups 1 and 2 were allowed to review previous MRI studies when available, but only the limited MRI sequences on the prior study could be reviewed when formulating an impression. Readers in both groups were not permitted to review post-contrast, MRA, or MRV images, if these were acquired.In order to eliminate recall bias, the assignment of MRI cases to each neuroradiologist was reviewed. If any case had been previously reviewed by the neuroradiologist to which the case was assigned, then the case was reassigned to an additional neuroradiologist (RGG) who reviewed the case with the same instructions.We designated findings and discrepancies as major if they were likely to have a significant impact on patient management. Level 1 findings were designated as those requiring immediate communication at our institution: acute infarction, intracranial hemorrhage and unexpected mass lesion. The following imaging findings were also designated as major: subacute infarctions; posterior reversible encephalopathy syndrome (PRES); vascular malformations; malformations of cortical development ; hydrocephalus; parenchymal lesions suspicious for infection, inflammatory disease, primary or secondary CNS neoplasm; and other mass lesions. Abnormalities of the major intracranial flow voids including arterial dissection, venous sinus thrombosis and intra-arterial thrombus were also considered major abnormalities. Acute/subacute infarctions required observation of decreased diffusivity in DWI sequences. Microhemorrhages  were considered potentially important findings. Other typically incidental findings were designated as non-major.A descriptive analysis of the data from groups 1 and 2 was performed.Out of a total of ∼3750 brain MRI studies, 998 brain MRI studies met our eligibility criteria. The first 497 studies were placed in group 1, and the remaining 499 in group 2.In group 1, 84 major findings were identified  . There wThere were 3 vascular abnormalities that were not identified on the abbreviated sequences; 2 cases of middle cerebral artery branch (MCA) thrombus in patients with concurrent acute infarctions and a case of transverse sinus thrombosis. The cases of MCA branch occlusion were not visualized on the 3-sequence series, although the acute infarctions in the MCA distribution were identified. The group 1 reader who did not identify the venous thrombosis reported that they would not have seen this lesion without an MR venogram.In group 1, there were 19 cases that had evidence of probable microhemorrhage (3.8%). Out of these 19 cases, 15 were not identified by group 1 readers on the abbreviated MR sequences (77.8%) .In the group 2 evaluation, there were 117 major findings in the 499 MR studies 23.4%, . ExampleSince the introduction of MRI for the clinical evaluation of brain pathology, a large number of pulse sequences have been developed, and those with the highest clinical value have been extensively evaluated. However, there is little documented research on the optimal set of MRI sequences for the evaluation of patients with specific clinical presentations. In this evaluation of MRI used in patients with a new neurological complaint, we found that a limited-sequence imaging protocol can identify nearly all significant brain abnormalities with the major exceptions of small, contrast-enhancing lesions and vascular lesions that are revealed by MR angiography. The 4-sequence protocol was superior to the 3-sequence protocol due to improved detection of probable microhemorrhages, and we conclude that it is sufficiently robust for the routine evaluation of patients who have new neurological symptoms and do not have a clear clinical indication for the use of contrast  or direct evaluation of the cerebral vasculature (e.g. vascular risk factors with high suspicion for acute ischemia).The T2-weighted FLAIR sequence has been shown to be the single most sensitive sequence for the detection of brain pathology Cerebral microhemorrhages are a common finding, but have indeterminate prognostic and therapeutic significance The use of the 3-sequence protocol resulted in lack of detection of 25% of major abnormalities, with over half of these being enhancing lesions. An improved detection rate was manifest with the 4-sequence protocol, with a lack of detection of 16% of major findings, but 90% of these were enhancing lesions. Enhancing lesions included meningiomas, active inflammatory/demyelinating lesions, metastases and probable primary brain tumors, as well as indeterminate punctate areas of parenchymal enhancement. Our findings suggest that intravenous gadolinium-based contrast should be administered in patients with a history of malignancy or suspected demyelinating/inflammatory disorders.Several significant vascular lesions were not identified on the 3- or 4-sequence protocols, even upon revisiting the study, but were identified on MRA or MRV. Two of the missed vascular abnormalities in group 1 were occlusions in MCA branches. In cases where arterial dissection, vascular thrombosis or occlusion is clinically suspected, a vascular imaging sequence such as an MRA or MRV may add considerable value .One case of cortical dysplasia in group 1 was not initially identified, but the dysplasia was discernible in retrospect . The finThe limited 4-sequence MRI protocol should increase efficiency and reduce scan length. The amount of time saved using the 4-sequence protocol is difficult to precisely calculate due to the variable pre-scan preparation times; however, we estimate that the time saved ranges from 5 to 25 minutes per study compared to our standard protocols. Further efficiencies in study acquisition may be possible through optimization of the limited sequence parameters. Also, substantial time savings in the interpretation of the 4-sequence MRI studies are expected since there will be fewer images per study for the neuroradiologist to review.The 4-sequence MRI protocol will only be efficient and of value if the limited sequences allow the neuroradiologist to make a correct diagnosis with a limited rate of patient recall for additional sequences. The data in our study suggest that the patient recall rate will be very low and on the order of “misses” using the standard protocol.A proper review of the patient's history should allow the neuroradiologist to tailor the MRI sequences to be performed for any given indication. The limited 4-sequence MRI screening protocol is only appropriate for the patient with new neurological complaints without an existing diagnosis. For example, a patient with known metastatic cancer will receive a contrast-enhanced MRI study. For patients undergoing the limited screening protocol, unexpected findings may be detected that warrant work-up with additional MR sequences. In these cases, we envision a hybrid solution. A large number of cases are monitored in ‘real-time’ by the neuroradiologist, which allows for immediate tailoring of the examination based on the findings on the limited sequences. Current technology even permits the neuroradiologist to remotely monitor cases and tailor them off-site. However, there will be some cases that cannot be monitored and such patients may require additional sequences to be performed at another time.This study has several limitations. It has a retrospective design, although effort was made to minimize bias by analyzing consecutive cases. Despite our diligent attempt to minimize bias, results should be interpreted with caution as they have not yet been validated in a prospective fashion or using active clinical cases. A prospective study is currently being designed to confirm the findings described here. An additional limitation is that the impressions from each original MRI interpretation were used as the reference standard. There may have been findings on brain MRI that were mentioned only in the body of a report and not in the impression. This most notably occurred in patients with probable microhemorrhages. Another limitation is that the original MRI interpretations were performed in the true clinical setting where the neuroradiologist may have been interrupted or distracted by phone calls, teaching and consultations, which is different than the clinical research conditions under which the group 1 and 2 neuroradiologists reviewed the cases. However, the group 1 and 2 neuroradiologsists interpreted their cases using the same PACS workstations in the same clinical work areas as those of the original interpretations and, therefore, were subject to similar disturbances and distractions. In addition, for the original clinical interpretations, the readers also had access to the full medical record that is readily accessible at the workstation, as well as the ability to contact the referring the physician for further information. Thus the reading of the limited sequences was more restricted than the original reading, thus providing an added measure of assurance on the validity of the results. The heterogeneity of our institutions MRI scanners, which had different manufacturers and field strengths, may be considered as another limitation. Bearing that in mind, the national installed base of clinical magnets has far greater heterogeneity than our institutions and one might argue that these studies cannot reflect the diversity of clinical practice. Additionally, this paper describes an effort to limit the number of sequences required for routine performance and is not making any comment with respect to interpretation times. Finally, the readers were primarily experienced neuroradiologists, which is not necessarily true for all those interpreting brain MRIs. We have not attempted to validate error rates utilizing this protocol amongst general radiologists or other specialists.A four-sequence MRI protocol that includes sagittal T1 weighted images, axial T2-weighted FLAIR images, axial diffusion-weighted images, and T2*-weighted images reliably detects nearly all non-enhancing brain MRI findings in patients without a known history of a neurologic disease undergoing evaluation for a new neurologic complaint. If a patient has persistent unexplained symptoms, an additional MRI study including post-contrast and high-resolution imaging should reveal abnormalities characterized by blood brain barrier breakdown or small structural lesions. Clinical information suggesting a vascular lesion should be evaluated using an angiographic or venographic sequence.


The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical. In mammals, light has a strong impact on daily activity rhythms by synchronizing an organism’s internal clock with rhythms in the external environment  and through more acute effects on general activity (a phenomenon known as masking). While entraining effects of light on the internal clock are very similar in diurnal and nocturnal species (reviewed in Many brain areas such as the suprachiasmatic nucleus (SCN), lateral geniculate nucleus (LGN), pretectum, and superior colliculus (SC) are innervated by ipRGCs in nocturnal mice, as indicated by studies of transgenic models . AnotherArvicanthis niloticus). These animals are native to sub-Saharan Africa and are diurnal both in the field and in the laboratory (A. ansorgei). Although several characteristics of these cells are similar to those reported in nocturnal rodents, some differences exist in the firing patterns of a select subtype of ipRGCs  cycle (lights on at 06:00 h) unless otherwise indicated. All experiments were performed in accordance with guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Michigan State University Institutional Animal Care and Use Committee. All efforts were made to minimize the number of animals used in these experiments.Adult male Nile grass rats from a breeding colony maintained at Michigan State University were used in this study. Animals were housed in plexiglass cages (34 cm × 28 cm × 17 cm) with access to food  and water n = 3) or unilaterally  enucleated or they underwent sham surgery (n = 3). For enucleations, the eye was held with forceps, the optic nerve and blood vessels were severed and the eye was removed. Absorbable gelatin was inserted into the orbit and the eyelid was sealed with Vetbond . Control grass rats were anesthetized but neither eye was removed. All animals were perfusion fixed 14 days after surgery and brains were collected and processed for visualization of PACAP (see below).Nine Nile grass rats were anesthetized with isoflurane and were either bilaterally  of CT-β conjugated to Alexa Fluor 488  in the right eye and Alexa Fluor 594  in the left; both were purchased from Molecular Probes  and were dissolved in 2% dimethyl sulfoxide (DMSO) in 0.9% saline vehicle. Seven days following surgery, grass rats were perfusion fixed (see below).We collected brains from the animals described above and both retinas from three other animals maintained in a 12:12 LD cycle and four animals that were kept in constant darkness (DD) for 5 days prior to sacrifice. All of these animals were anesthetized with an intraperitoneal injection of either sodium pentobarbital  or urethane  and transcardially perfused with 0.01 M phosphate buffered saline  followed by Stefanini’s fixative , or 4% paraformaldehyde . Brains and eyes were extracted, postfixed in Stefanini’s fixative or 4% paraformaldehyde for 12–18 h, then immersed for at least 48 h in 20% sucrose solution in 0.1 M phosphate buffer (PB) and kept at 4°C. Brains were cut into coronal sections (30 μm) using a microtome [for single-label PACAP immunohistochemistry (IHC)] or a cryostat (for brains labeled with fluorescent agents). Three alternating series of sections were collected from each brain and were stored in cryoprotectant at -20°C until further processing. Retinas were orientated and dissection was performed by first removing the cornea, followed by removal of the lens. Hereafter, four cuts were made to mark the superior, nasal, inferior, and temporal quadrant and for orientation a small cut was made in the superior quadrant. The eye was then held in place by needles and the vitreous was gently removed with forceps and filter paper. After gentle dissection along the ora serrata and cut of the optic nerve, the retina was removed and kept in cryoprotectant solution , 30% ethylene glycol, 0.05 M sodium phosphate buffer, pH 7.2) for better conservation, and thereafter stored at –20°C until immunohistochemically processed.A mouse monoclonal anti-PACAP antibody  recognizing the epitope between amino acids 6–16 was used for both the brain and retinal tissue; this antibody has equal affinity for PACAP-27 and PACAP-38  and show202 in 0.01 M PBS for 10 min, blocked with 5% normal donkey serum for 20 min, and then incubated with the PACAP antibody for 72 h at 4°C. Retinas were then incubated in the secondary antibody, biotinylated donkey anti-mouse  over night at 4°C. Next, tissue was incubated in avidin-biotin complex (ABC) solution (0.9% each of avidin and biotin solutions) for 30 min, biotinylated tyramide  for 1 h, and finally Alexa Fluor 488-conjugated streptavidin . Hereafter, tissue was placed in 1% H202 in 0.01 M PBS for 15 min, washed in PBS and incubated in the mixture of N- and C-terminal melanopsin antibodies for another 72 h at 4°C. After a rinse, the retinas were then incubated in Envision reagent  overnight and visualized by tyramide conjugated Alexa Fluor 594 .To label both PACAP and melanopsin in the retina, double label IF was used. Tissue was rinsed with 0.25% Triton-X-100 (TX) in 0.01 M PBS between all steps of the procedure, and all incubations included 0.25% TX and 0.25% bovine serum albumin (BSA). All rinses and incubations occurred at room temperature, unless otherwise noted. Retinas were first treated with antigen retrieval (AR) solution in citrate buffer  at 80°C for 1.5 h. Next, tissue was pre-incubated with 1% HThe IHC procedure used for single labeling of PACAP in the brain was like that used for PACAP in the retina with the following exceptions: the AR step was not included, incubation with the PACAP antibody was for 48 h, the concentration of the biotinylated tyramide in which sections were incubated was 1:100, and after the 30 min incubation in ABC solution, sections were rinsed two times in 0.25% TX in 0.01 M PBS, then placed in Tris buffer (pH 7.6) for 10 min, preincubated in 0.06% diaminobenzidine  in Tris buffer for 30 s and reacted with 0.01% hydrogen peroxidase for 2.5 min.Immunofluorescence (IF) was used to label PACAP in one series of brain sections from each of the CT-β injected animals. The procedure was similar to that used for PACAP + melanopsin IF, except that incubation of the antibodies were somewhat shorter (48 h in the PACAP antibody and 1 h in the secondary), and Cy5-conjugated streptavidin  was used instead of Alexa Fluor 488-conjugated streptavidin.2 and represented both the central and peripheral retina. The retina was photographed with an iMIC confocal microscope  using the wide field camera and 10× objective. Photographs were taken of each part of retina and stitched together using the LA Stitch plug-in in Fiji software  to create an image of the entire retina. Each of these was then analyzed using the cell counter plug-in Fiji to mark and count which cells contained only PACAP, only melanopsin, or both. Counts of melanopsin-stained cells were performed on retinas from animals either maintained in LD or kept in DD for 5 days, since a previous report in Brown Norwegian rats indicated that melanopsin expression is highest in animals maintained in constant darkness (Z-axis = 40 μm) of the entire retina covering the ganglion cell layer (GCL) until the inner nuclear cell layer . After stitching all Z-stacks together using the LA Stitch plug-in in Fiji software , melanopsin cell subtypes, as defined previously in mouse retina  attached to a Zeiss light microscope , while fluorescent images were obtained using an iMIC confocal microscope  equipped with appropriate filter settings for detecting DAPI, Cy2/Alexa Fluor 488, Texas Red/Alexa Fluor 561/594, and Cy5. Determination of whether PACAP and melanopsin were present in a single cell was done with a co-localization plug-in module in ImageJ/Fiji software  in which the points of two 8-bit images with both antigens appeared white . Pixels were considered to reflect co-localization of the antigens if their intensities were higher than the threshold of their respective channels (we used a threshold set at 50–100 depending on the background noise) and if the ratio of their intensity was higher than the ratio setting value (we used the default set at 50%). Melanopsin/PACAP cell counts were performed on areas from six pieces of the same retina in which cells were stained well for both PACAP and melanopsin; areas in which one or both immunoreactions were insufficient were excluded. The sizes of the pieces ranged from 0.65 to 8.7 mmdarkness . The entFigure 1). Melanopsin was located mainly in the cell membrane of the soma and dendritic processes but in animals kept in constant darkness melanopsin was also found in the membrane of axons projecting to the optic nerve . As previously reported in mice . We were unable to identify melanopsin cell types as M4 or M5 in the grass rat retina. Whereas M1 and M3 cells , as well as M2 cells  were scattered relatively evenly across the entire retina, displaced M1 cells  were located primarily in superior regions of the retina . Interestingly, a large number of melanopsin-expressing cells were found in of the superior, nasal, and temporal periphery of the grass rat retina  as recently described in the rat (2) were present whereas in animals housed in constant darkness, 1,402 ± 52 melanopsin cells (32.7 ± 1.3 cell/mm2) were counted; this difference was not statistically significant [t(3) = 2.165, p = 0.12). However, the dendritic network appeared to be denser and axonal staining of melanopsin was present in animals housed in constant darkness.Melanopsin expression was examined in flat mount retinas from animal housed in a 12:12 LD cycle and from animals housed in constant darkness due to previous reports in rats showing a slight increase in melanopsin protein expression during prolonged periods in constant darkness . Melanop the rat  and mous the rat . These cFigures 2 and 3); this percentage ranged from 84 to 94 across the different pieces of retina. PACAP was seen in all subtypes of melanopsin cells. A very small number of cells  containing only PACAP  were counted. None of the six pieces of the retina had a melanopsin cell density that was below the average density found when counting the total number of melanopsin cells. This finding in the grass rat retina was similar to that of the monkey retina . Since we found some variation between the numbers of melanopsin/PACAP containing cells in the different pieces of retina we cannot exclude the possibility that some melanopsin cells don’t express PACAP (and vice versa) but more likely this finding is due to low levels of expression of PACAP or melanopsin in some cell types.A total of 633 cells containing melanopsin were counted in six pieces of the same retina. Of these, 555 (87.7%) co-stored PACAP , while in the mid-caudal portions of the SCN, PACAP labeling was present across the nucleus . PACAP-ir fibers were greatly reduced in bilaterally enucleated grass rats compared to shams throughout the rostrocaudal extent of the SCN . In unilaterally enucleated grass rats PACAP fiber labeling was reduced but still present ; the reduction was similar in the SCN ipsilateral and contralateral to the eye that had been removed, indicating that PACAP fibers from the retina have bilateral projections to the SCN. This was confirmed by the bilateral tracing experiments, which also show that the majority of PACAP-ir nerve fibers in the SCN originate from the retina .In the rostral SCN of sham grass rats, PACAP-ir fibers were more ventrally located . Most of these fibers come from the contralateral eye , as previously shown in these animals ; this pattern was similar to that of melanopsin cell projections to this area in mice  and were only present in the dLGN contralateral to the remaining eye in unilaterally enucleated animals  indicating that melanopsin/PACAP projections from the retina to this region are completely crossed. Very few PACAP-ir fibers were observed in mid-caudal dLGN ; however, those seen were overlaid with CT-β-labeled retinal fibers from the contralateral eye .The bilateral retinal tracing demonstrates a distinct pattern of projections from the eye to this part of the grass rat brain . Their distribution in this region overlapped to some extent with that of CT-β-labeled retinal fibers emanating from the ipsilateral eye . A few PACAP-ir cells were present in the vLGN. These cells were in the outer boundaries  of the vLGN and were relatively sparse in the central region.Relatively few PACAP-ir fibers were found in the ventral portion of the vLGN . Although PACAP-ir labeling was not reduced in bilaterally enucleated grass rats in the most rostral portion of the IGL  PACAP-ir in the mid-caudal region of the IGL was substantially reduced after removal of both of the eyes . In unilaterally enucleated animals PACAP-ir was diminished slightly more on the side of the brain contralateral to the eye that had been removed , indicating that many of these fibers are crossed; however, many fibers were still present on both sides of the IGL, indicating that PACAP-ir projections from the retina to the IGL are bilateral. This was supported using bilateral tracing, which demonstrated a high degree of overlap between the retinal tracers from the two eyes with PACAP fibers in the IGL . Similar to the vLGN, a few PACAP-ir cells were found within the IGL .Pituitary adenylate cyclase-activating polypeptide-immunoreactive fibers densely innervated the IGL and spanned across its full rostrocaudal extent . Although the rostrocaudal extent of the grass rat OPT, as defined by retinal projections, is extensive . Most PACAP-ir fibers in the OPT originated from the retina, as bilateral enucleation greatly reduced their density . PACAP-ir fibers were observed in both the left and right OPT in unilaterally enucleated grass rats, but more were present in the OPT ipsilateral to the eye that had been removed, indicating that most fibers are crossed . This observation was supported by the high degree of overlap between CT-β positive retinal fibers and PACAP-ir fibers within the OPT , especially at its most rostral pole . A distinct distribution of ipsilateral and contralateral innervation was revealed by the bilateral tracer injections which demonstrated that contralateral projections target the central and ventral OPT, while input from the ipsilateral eye is concentrated in the peripheral part of the dorsal region of the OPT . Most prominent co-localization between PACAP and Ct-β was found in the central and ventral part of the OPT.Pituitary adenylate cyclase-activating polypeptide-immunoreactive fibers within the pretectum were concentrated in the olivary pretectal nucleus . However, many PACAP-ir fibers in the PLi did not overlap with the retinal tracers, indicating that many of the PACAP fiber within this area do not originate in the retina.Another pretectal structure that contained PACAP-ir fibers was the posterior limitans (PLi). Although PACAP-ir did not seem to be reduced in enucleated grass rats, overlap between PACAP-ir and the retinal tracer from the contralateral eye indicate that a few of these fibers originate from the retina  but they were absent in bilaterally enucleated ones . In unilaterally enucleated animals PACAP-ir labeled fibers were denser on the side contralateral to the remaining eye and virtually eliminated on the ipsilateral side . Similarly, there was a high degree of overlap between PACAP-ir fibers and CT-β-labeled retinal fibers from the contralateral eye , again demonstrating the crossed nature of PACAP-containing retinal fibers in this region. As found in other rodents, PACAP-expressing neurons were found in the deep layers of the SC (not shown).A few intensely labeled fibers were found in the SC of intact grass rats (p = 0.12). This trend, however, followed the same pattern as that seen with melanopsin-immunoreactivity in brown Norwegian rats ; a larger number of ipRGCs are observed in the superior retina of nocturnal rats . Similar clusters are reported in the peripheral region of either the superior . The number of RGCs co-storing PACAP and melanopsin was slightly lower compared to that previously reported in the rat, hamster, monkey, and human . These areas are all known to receive such input in other species . The few non-retinal PACAP-ir fibers within the SCN may come from the IGL or vLGN, as there are weakly stained PACAP-ir neurons in both areas in grass rats . Such cells have not been reported in Wistar rats . Data derived from lesion studies of nocturnal rodents led Intrinsically photosensitive retinal ganglion cells projections to the SCN of nocturnal mice carry information about light that comes both from its effect on the melanopsin protein and from cone/rod-driven pathways that converge on the ipRGCs and this pathway mediates the entraining effects of light on circadian rhythms . The ipRFigures 8). In this respect, Nile grass rats are very different from nocturnal rodents, in which both the dLGN and vLGN are heavily innervated by ipRGCs , regions that receive input from ipRGCs in other species as well . Interestingly, we only saw these ipRGC fibers in the most rostral portion of the OPT, whereas in nocturnal mice they are present across its full rostrocaudal extent (unpublished data). Further, effects of light on the OPT are quite different in Nile grass rats and nocturnal mice; specifically, light induces an increase in FOS in the OPT of the former species and a decrease the latter . In mice, both M1 and non-M1 ipRGCs project to the SC . However, some interesting differences were apparent. For example, the ventral and dorsal LGN appear to receive less input from ipRGCs in Nile grass rats than in nocturnal murid rodents. Furthermore, whereas ipRGCs project to full rostrocaudal extent of the OPT in other species, the caudal OPT does not seem to receive such input in grass rats. Finally, it should be noted that there might be differences within target structures identified here with respect to which cell populations receive direct input from the ipRGCs. Thus, future studies are needed to test the hypothesis that differences in patterns of connectivity between ipRGCs and their targets contribute to differences between diurnal and nocturnal species with respect to their masking responses to light.In conclusion, ipRGCs, as defined by the presence of melanopsin, contain PACAP in the diurnal Nile grass rat, as they do in nocturnal rodents (hamsters: All authors take responsibly for the accuracy and integrity of the described work. The concept and experimental design were performed by JL, LS, and JH. All authors contributed in some way to the acquisition and analysis of the data. The initial drafting of the work was done by JL and all authors were involved in subsequent revisions of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malnutrition is frequent among older people and is associated with morbi-mortality.The aim of the study is to assess the effectiveness of a multifactorial and multidisciplinary intervention in the nutritional status among the elderly.Randomized, single-blind, parallel-group, clinical trial conducted from January 2009 to December 2010 in seven primary health care centers in Baix Llobregat (Barcelona). Of 696 referred people, born in 1924, 328 subjects were randomized to an intervention group or a control group. The intervention model used an algorithm and was multifaceted for both the patients and their primary care providers. The main outcome was improvement in nutritional status assessed by Mini Nutritional Assessment (MNA). Data analyses were done by intention-to-treat.Two-year assessment was completed for 127 patients (77.4%) in the intervention group and 98 patients (59.7%) in the control group. In the adjusted linear mixed models for MNA, intervention showed no significant effect during all follow-up period with −0.21 . In subjects with nutritional risk (MNA ≤ 23.5 / 30) existed a tendency towards improvement in MNA score 1.13  after 2 years.A universal multifactorial assessment and target intervention over a two year period in subjects at nutritional risk showed a tendency to improve nutrition but not in the rest of community-dwelling studied subjects. Cognitive impairment was an independent factor strongly associated with a decline in nutritional status.NCT01141166.The clinical trial is registered as part of a US National Institutes of Health Clinical Trial: The online version of this article (doi:10.1186/s12877-015-0033-0) contains supplementary material, which is available to authorized users. Over past decades, the oldest group (aged over 80) has been the most rapidly growing population segment, and is expected to reach 10% in developed countries by 2050 , althougIn the literature, the effectiveness of nutrition interventions in older people has received growing attention. A recent Cochrane review on food supplements showed that nutritional supplementation produced a small weight gain in older people with no evidence of the potential benefits for an improvement in functional status . PositivOnly a few randomized clinical trials -13 have This study was a randomized single-blind, parallel-group, clinical trial (registration number NCT01141166) of a multifactorial malnutrition intervention in community-dwelling elderly people conducted from January 2009 to December 2010. The design of the study and baseline data has been published in detail elsewhere [All community-dwelling inhabitants born in 1924 and registered at one of seven primary healthcare centres in Baix Llobregat  were contacted. The combined population served by these healthcare teams includes approximately 210,000 inhabitants of a total of 800,000 inhabitants of the Baix Llobregat area (17% being people older than 65). The seven voluntary health care centers involved in other elderly assessments were in the same geographical area and they had similar data regarding immigration percentage (11%) or population served (70%).The only exclusion criterion was institutionalization . We did not use exclusion criteria for diseases or cognitive impairment. Research assistants contacted potential participants (by telephone or letter), and those who had no exclusion criteria were asked to participate. All subjects who agreed to participate signed informed consent forms before the study started. People unable to give informed consent were included if they had a relative caregiver who gave informed consent. There were no differences among respondents and non-responders in terms of gender, health care center, or physician in charge.Data collection was performed of all subjects according to medical records and interviews in primary care services or at home if the subjects were not ambulatory . It was done by a health assistant , at baseline, and at 12 and 24 months of follow-up.Gerontology assessment included socio-demographic data such as gender, marital status, education level, presence of caregiver and whether they lived alone. The evaluation of co-morbidity placed special emphasis on diagnosed hypertension, diabetes mellitus, dyslipidemia, ischemic cardiomyopathy, heart failure, stroke diagnosis, atrial fibrillation, dementia, Parkinson’s disease, and previous diagnosis of anaemia. We used the Charlson Index to assess global co-morbidity, which has a minimum score of 0 for a healthy individual and a maximum score of 37 for high co-morbidity . In addiThe assessment included sensory status (near vision measured by the Jaeger test and hearing ability measured by the whisper test) . FunctioOnce the baseline questionnaire had been administered, subjects were randomized to the intervention or control group using a computer-generated randomization table. The allocation was double-blind . The researcher who prepared the randomization lists and allocated subjects to study groups had no contact with the study subjects.Of the 696 subjects screened for the study, 142 did not meet inclusion criteria and 67 died before they were contacted. Of the 487 eligible subjects, 328 (67.4%) were randomized. The loss before randomization was 84 subjects who declined to participate and 75 subjects who could not be contacted . Figure Subjects in the intervention group received a community based multifactorial program that links participants to existing medical care and service networks. The intervention was aimed at malnutrition risk factors, and consists of a treatment plan that used a specific algorithm and was multifaceted for both the patients and their primary care provider’s. Additional file Participants were advised to contact their primary physician within 1 month to review these results, recommendations, and referrals. The family physician of each participant was mailed after the examination to discuss referrals to medical specialists, medication changes, and follow-up.The algorithm in the intervention participants evaluated chronic prescription. Special emphasis was placed on the importance of the number of drugs, the progressive discontinuation of benzodiazepines and the use of vitamin supplementation. Subjects were referred to an ophthalmologist when the corrected monocular worst near vision was less than 0.5/1 decimals on the Jaeger chart (<5 Jaeger). If there was any visual field impairment, the patient was also invited to have home modifications to improve visibility using lighting modifications. Participants received nutritional advice on dietary matters and referral to physical therapists for assessment. Information was also reinforced with printed sheets of standard dietary recommendations for the elderly.The algorithm generated recommendations also for corrected auditory impairment, gait disorders, functional or cognitive decline, home environment barriers and social risk and referral to other medical specialists was initiated when deemed necessary .After the initial program session, a health assistant involved in the research intervention assessed its adherence and interim results with a follow-up visit or telephone call every 3 months during the first and second year. The researchers listed any actions that participants reported that they had self-initiated during and after the first session, as well as actions and recommendations that were discussed during the home visit. The additional purpose was to answer questions and to encourage adherence to nutritional advice, physical therapy and exercise, and to other recommendations.During the second year, two specific interventions were offered: nutritional and rehabilitation assessment. Subjects at nutritional risk (MNA ≤ 23.5/30) received three one-hour individual meetings with a dietician  who developed a plan for individualized nutrition. The content of the meeting included food diary analysis, advice on dietary adaptation to address the most common nutritional problems, recommendations regarding basic cooking techniques, adaptation of textures, or nutritional supplements for patients in need of extra calories. At the end of the sessions, participants received printed information for use at home. On participants with cognitive impairment, the role that played the caregiver was important. They received general information about nutrition, nutritional needs of patients with dementia  or other problems related with nutrition in order to assure adherence to the recommendations to the recommendations given. Subjects with one or more falls and no cognition impairment (MEC > 19/35) and ambulation preserved received, over 6 months, four 90-minute sessions with a physiotherapist coordinated by a specialist in Rehabilitation .To standardize the procedure, the intervention research team received specific training in multidisciplinary geriatric assessment, from a geriatric specialist with experience in educating health professionals for 2-hour sessions a week for 3 months.The control participants received usual health care which includes a comprehensive general medical consultation when they had a health complaint, limited by a short physician consultation time (7 minutes).The primary outcome measure was to assess the effectiveness of nutritional intervention in nutritional status evaluated by the MNA. Secondary outcomes evaluated were the changes over time of diagnosis of anaemia, functional status, analytical parameters and the number of such incidences . Nutritional status and secondary outcomes were ascertained during the annual assessment and self-reports using the monthly calendar  or also by medical record data. The health assistant conducted the 24-month follow-up assessment. In the case of loss of follow-up because of death, the date of death was documented.Adherence to recommendations was also monitored, using quarterly visits or telephone calls made by the health assistant. A recommendation was adhered to if at least 70% of the session’s program was completed at any time in the 24-month period. To corroborate adherence information, the reports were verified using the primary care medical records .For the sample size estimation 30% of prevalence of malnutrition and an expected reduction of 15% was assumed (Relative Risk = 0.5). Assuming 0.10 alpha error and beta error of 0.20 in a unilateral test, the necessary sample size was estimated as 164 individuals both in the control and intervention group. A 50% of replacement was assumed . For the sample size the estimation GRANMO 5.1 was used.Categorical variables are shown as frequencies and percentages, while continuous variables are presented as means and standard deviations or as medians and interquartile range, depending on normality assumptions . In the bivariate analysis for baseline characteristics either the Chi-square or Fisher’s exact test was used to compare categorical variables between the control and intervention group. The Student’s t-test or the Mann–Whitney test, depending on the normality of the variable, was applied to compare continuous variables. As in other studies, we defined two groups: those who were well nourished MNA > 24) and those at risk of being undernourished, which included people at risk of malnutrition (MNA 23.3–17) and those who were malnourished (MNA < 17) [ and thosThe primary outcome for malnutrition was analyzed as the evolution of the values of MNA for all follow-up periods adjusting linear mixed models, comparing the control and intervention group. For all models the interaction of the intervention group and time period variable  was adjusted in order to take into account differences in MNA evolution between groups. Other models with baseline characteristics of the participants were adjusted. Secondary outcomes were compared between groups as mean differences for continuous variables and incidence for categorical ones on anaemia, functional, analytical and healthcare utilization for the evolution from baseline to 24-month follow-up.All analyses were carried out by intention to treat. The significance level was set at 0.05. All analysis was performed using R software .For the study 328 subjects were randomized. A total of 202 61.6%) were female, 62 (18.9%) had been educated for more than 6 years and 100 (30.5%) lived alone. Caregivers existed in 174 (53.0%) elderly people. The baseline characteristics of the two groups were similar in terms of age, sex, living alone, co-morbidity and most health-related variables. The geriatric assessment at baseline produced the following mean values: The median Charlson index was 1.00 (0–7) and 199 (61%) subjects was less than 2. The Jaeger test score for median near visual acuity was 5 (3–10), while auditory impairment was present in 124 (37.8%) individuals. In the evaluation of daily activities, the median BI was 95 (85–100) and there were 228 (70%) subjects with IB greater than or equal to 90 and 23 BI individuals scored < 60. The LI median value was 6 (4–8). For cognition, the MEC score was 28 , while  individuals scored MEC < 19. According to the MNA, 215 (65.50%) individuals had correct nutritional status, 101 (30.8%) were at nutritional risk of malnutrition, and 12 (3.7%) individuals had malnutrition at baseline. The median score on Gijón’s scale was 10 (8–11) and 172 (52.4%) subjects was in social risk > 10. The EQ-VAS value for median quality of life was 60 (50–75). The Tinetti scale median score was 8 (5–9). During the previous year, a total of 93 individuals (28.4%) had had at least one fall while 25 subjects (7.6%) had ≥ 2 falls in the past year Table .6% were The evolution of the MNA by group and follow-up period is shown in Figure Subjects who received specific nutritional intervention (47 participants with MNA ≤ 23.5) showed a non-statistical increase in MNA score with 95% confidence interval of 1.13  after one year follow-up (from 12 months to 24 months follow-up). A non-statistical significant decrease in MNA score  was found in control group participants from first to second year follow-up Table Evolution for secondary outcomes from baseline to 24 months of follow-up Analyses of secondary outcomes yielded a significant difference in longitudinal changes in the control group respect to the intervention group in LI (p = 0.01). The BI, health care utilization and analytical parameters did not show a difference at the 24-month follow-up, although the intervention group showed a significant tendency of a lesser decline in haemoglobin levels compared to patients in the control group .Table The main result of the present study is that a multifactorial assessment with individual intervention in the oldest old people living in a community-dwelling has not provided evidence of effectiveness in nutritional status. However after receiving individualized nutritional support, subjects included at baseline in the subgroup of “at malnutrition risk” showed improvement in the MNA score. To remark that we found that cognitive impairment is an independent factor strongly associated with a decline in nutritional status.The Octabaix study included a combined community –dwelling population of oldest old with comorbidities and cognitive impairment. The results of this study showed a group of successful agers with an acceptable health, functionality preserved, good cognition and low comorbidity as published on others studies. Moreover health status of patients living in the community is better than older people living in Nursing homes or Long Term Care Units ,26.Nevertheless a Cochrane revision suggest the effectiveness in improving nutritional status in the elderly , there iIn fact, in the present study during the follow up no significant differences in nutritional status were found during the follow up; nonetheless, subjects at malnutrition risk showed a tendency improvement in nutritional status at the end of the second year. These findings are consistent with similar studies in which no significant change in body composition and energy intake was found after individual nutrition counseling and physical exercise in community-dwelling frail elderly people aged 75 and older ,28. In cAnother reason for the limited effectiveness of this program is the capacity of our national health care system. The education support training of general practitioners is a key to learning about the importance of good care for the health of the elderly . DespiteThe third explanation is a lower level of education and subjective health perceptions in the intervention group at baseline. These findings contrast with those described by Kaplan et al. which identified goal setting, motivation, interaction with health care providers and self -reported health perceptions as some of the effective features of nutrition education interventions among older adult participants ,30. TherThe fourth reason could be that subjects who left the study were often sicker and frailer than those who remained with more co-morbidity and poly-pharmacy. Since the drop-out was higher in the controls than in the intervention arm, then if the sickest and most frail were more often lost from the control group compared to the intervention arm, the remaining controls would have a tendency to be healthier than the intervention group, so the control subjects may be less likely to have developed malnutrition and other adverse events than the intervention group, due to differential rates of loss of the highest at-risk participants.Another important reason to take into account could be the contamination effect of the control group. Some of the health professionals at the centers involved in the trial had patients in both the intervention and the control group in their practices. The study design did not permit them to know the group allocation of their patients, but when participants came in asking for a medication review or for referral to certain kinds of specialist consultation, practitioners could guess to which arm of the study they belonged. Also there could have been communication among participants. However, this probably would have had a negligible effect ,32.Nutritional intervention has been used by other groups and in other conditions; so many studies evaluated and reported a correlation between risk of malnutrition and cognitive impairment ,34. Our Functional decline is often multifactorial in older adults and multifactorial intervention strategies might assist in the prevention or postponement of disability. Previous research showed that lower functional status in old nursing home residents may respond better to protein intake , and subThe MNA scores have been found to be significantly correlated to nutritional parameters in frail older persons . In our Health care use during the study period was compared between the groups. In the literature the decrease in costs of hospitalization related by nutritional status is not proved ,11, despThe strengths of this study are the external generalizability and applicability of the results due to the inclusion of all of oldest-old residents of similar aged registered in the national health care system in one area, regardless of their somatic and cognitive comorbidities.Among the main constraints that should be taken into account, there is a lack of evaluation of the psychological aspects such as depression. Another limitation was that the dietary energy and protein intake were not calculated at the baseline and follow-up, we accepted that MNA score is more representative of the real situation in primary health care, whilst another limitation concerns the high loss of follow-up in this study as in other similar studies, but it resulted in a number of participants that is similar to the median sample of other trials .In conclusion the present study shows that individually multifaceted multidisciplinary interventions have not consistently shown evidence of their benefit in long-term nutritional status in a cohort of not selected elderly people living at home but, this study highlighted the positive effect on nutritional status in old people at risk of malnutrition. While the impacts of nutritional intervention as well as the best intervention model are still debatable in well-nourished community dwelling participants, there are studies showing that more intense nutritional counseling, specific nutrition intervention, including tailored diet strategies, and nutritional supplement improves dietary intake as well as positive outcomes in the nutritional status or nutrition-related outcomes in elderly at risk of malnutrition or undernutrition living in the community. Future longitudinal studies should emphasize preventive measures in the oldest old people living in a community-dwelling with good nutritional status to prevent undernutrition and specific dietary primary health care programs by medical practice and dietician in patients at risk of malnutrition or malnourished living at the community, and target groups are needed to identify the best treatment model, including new training protocols, new strategies regarding primary care providers, community programs and an investigation of the role of caregiver to improve the assessment of home risk in the most elderly.


ND) measured by positron emission tomography (PET) [11C]raclopride imaging correlated with severity of wounding . Furthermore, utilizing standardized cognitive function tests, we showed that impulsivity  and deficits in attentional set shifting  were correlated with increased severity of SIB . We also tested the efficacy of guanfacine, an α2A adrenergic agonist that acts to improve postsynaptic transmission of neuronal impulses, in reducing SIB. A subset of these animals were enrolled in a randomized experimenter-blinded study that demonstrated guanfacine decreased the severity of wounding in treated animals compared with vehicle-only-treated controls (P=0.043), with residual beneficial effects seen for several weeks after cessation of therapy. Animals with the highest severity of SIB that received guanfacine also showed the most significant improvement . The elevated PET BPND was likely due to low intrasynaptic DA, which in turn may have been improved by guanfacine. With underlying physiology potentially representative of the human condition and the ability to affect outcome measures of disease using pharmacotherapy, this model represents a unique opportunity to further our understanding of the biology and treatment of SIB in both animals and humans.Self-injurious behavior (SIB) is a common comorbidity of psychiatric disorders but there is a dearth of information about neurological mechanisms underlying the behavior, and few animal models exist. SIB in humans is characterized by any intentional self-directed behavior that leads to wounds, whereas in macaques it is not always accompanied by wounds. We describe a cohort of rhesus macaques displaying SIB as adults, in which changes within the central nervous system were associated with the SIB. In these macaques, increases in central nervous system striatal dopamine (DA) receptor binding (BP Alterations in the brain dopaminergic system, with its role in reward-driven learning5 and working memory,6 is one proposed mechanism by which early experience and trauma could manifest as later psychiatric diseases and subsequent SIB.In humans, self-injurious behavior (SIB) is a high-impact manifestation of underlying psychiatric disorders, with outcomes ranging from social ostracism to severe physical injuries and/or death.8 In addition to difficulty with certain cognitive tasks, especially those associated with working memory, Lesch–Nyhan syndrome patients have been demonstrated to have decreased dopaminergic function,9 DA transporter,10 and dopaminergic nerve terminals and cell bodies.11 In rodent models, targeted disruption of the dopaminergic system by administration of methamphetamine or 6-hydroxy-DA has been used to model human SIB.13In humans, the most compelling case of alterations in dopamine (DA) contributing to SIB is in Lesch–Nyhan syndrome, a genetic disorder characterized by abnormal purine metabolism resulting in impaired cognitive development and function, as well as distinctive patterns of SIB.14 Among adult patients with a diagnosis of schizophrenia or borderline personality disorder, severity of SIB is positively correlated with errors on multiple cognitive function assessments.15 In addition to correlating with greater general cognitive impairment and decreased speech ability among children with autism, elevated severity of SIB is also associated with increased levels of impulsivity.16 Similarly, rhesus macaques with SIB have been demonstrated to exhibit cognitive alterations, including longer time to extinction of a learned behavior than controls.17The connection between altered cognitive function and SIB has long been established. The strong association between SIB and mental retardation has been documented by work beginning in the late 1970s.18 Nonhuman primates (NHPs) exhibiting SIB are more suitable because of their phylogenetic proximity to humans and resultant analogy in relevant behavioral repertoire, anatomy and physiology. SIB occurs sporadically in NHP housed in research settings, with reported prevalence ranging from 5 to 25% among individually housed rhesus macaques.21 SIB animals are usually identified by direct observation of the behavior or incidences of self-injurious wounds documented in the veterinary record.20 SIB in NHPs is defined by self-directed behavior including hair plucking, head banging and self-biting, each of which may or may not lead to tissue damage.19 Single housing at an early age, duration of single housing, number of blood draws/sedations, sex and nursery rearing have been document as risk factors associated with the development of SIB in NHPs.20 Whereas many characteristics of NHP SIB make them a potentially useful animal model, the sporadic nature and late onset of detectable behavior, which is frequently seen only after animals have already been assigned to unrelated studies, have historically made the formation of a suitable study cohort difficult. Nonetheless, reports of treatment for NHP SIB largely mirror the human literature, both in the specific treatments used and their mixed or inconsistent results.23Animal models hold the potential to further elucidate the pathobiology underlying SIB and its comorbid disorders and to serve as a means for testing novel therapies. Whereas rodent models of SIB have elucidated potentially related neurochemical changes, their deviation from the human condition in relevant behavioral manifestations, anatomy and physiology may limit their usefulness.24 Guanfacine, a preferential α2A receptor agonist originally used to treat hypertension, and more recently to treat ADHD, was reported in another case study to decrease wounding in two rhesus macaques and one baboon with SIB.25Adrenergic α2 agonists are one class of drug that has shown promise in both the human and NHP literature. In one case report, the α2 agonist clonidine was demonstrated to alleviate SIB with wounding in a young girl.Herein we describe a cohort of 19 rhesus macaque males, 7 years of age (YoA) that developed a high incidence of self-inflicted wounds. We hypothesized that the severity of SIB in these rhesus macaques would relate to measurable neurocognitive dysfunction similar to that described in humans with SIB, and that this dysfunction would develop before and relate to the clinical severity of wounds. We further hypothesized that pharmacological intervention using guanfacine, an α2A adrenergic agonist, would reduce both severity and frequency of wounding.Macaca mulatta), 7 YoA and weighing an average of 10.7 kg (range of 8.3–14.9 kg), were housed in compliance with the Animal Welfare Act Regulations and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals at Johns Hopkins University facilities that are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The animals were singly housed for the duration of the study to allow for administration of therapy and to ensure that any wounds present were self-inflicted. The animals were maintained on a 14-h light and 10-h dark schedule, fed a commercial macaque diet , given water ad libitum and provided with environmental enrichment  daily.Nineteen adult male rhesus macaques , intra-/extradimensional (ID/ED) and [11C]raclopride binding positron emission tomography (PET) were included in the retrospective analysis. For this same reason, coupled with the fact that previous studies have found some SIB ‘treatments' to actually exacerbate wounding,22 we wanted to include a small number of animals with no baseline wounds in our treatment arm of the study, thus we could ensure that guanfacine does not have a significant negative effect on such animals, such as might result from disinhibition. Of the 19 original animals in our cohort, six showed no baseline wounds despite documented histories of self-biting behavior. We randomly selected three of these six animals for inclusion in the guanfacine treatment arm of this study, and the other three animals were transferred to another study before beginning the treatment.Our chosen dependent measure for the purpose of this study was wound severity and distribution in the form of wound score (WS). Although we have demonstrated that for animals with wounds, this WS is related to biting behavior,27 they then had been provided access to oral cocaine and/or alcohol from ages 5.5 to 6.5 years (unpublished data). This experiment required that the animals be singly housed in cages, yet still allowing for visual and auditory contact, which began when the animals were between 458 and 660 days of age. The first clinical record of wounds secondary to SIB occurred when the animals were 6 YoA, with the prevalence of wounds and/or observed self-biting reaching 100% by 7 YoA. Importantly, there were no significant differences in wounding incidence or severity as a function of previous experimental treatment group .The animals had served in a study examining the effects of early-life administration of stimulant drugs and had been exposed to one of three treatment regimens  between the ages of 2 and 4.5 years;28 In cases where wounding is present, however, it is possible to measure the outcomes of SIB in terms of tissue trauma. Iwata et al.29 and Grace et al.30 have described using WSs and locations as a means of quantifying SIB in human cases where direct observation is difficult. We adapted this method to NHPs to obtain WSs, with consideration to clinically relevant differences in wound severity and distribution. We have previously shown that our WS system is associated with observed SIB behavior, and represents the severity and distribution of wounds in individual macaques.26Objective measurement of SIB can be quite difficult and labor intensive. Although 24-h direct observation is the ideal way to measure the behavior, in the majority of both human and NHP cases this is not possible owing to practical limitations of having observers present at all times and/or the high manpower costs of reviewing remotely obtained video. Previous reports examining SIB in monkeys have thus relied upon representative sampling for observations, but typically report relatively small samples .−1 ketamine HCl intramuscularly, and physical examinations were performed at 2-week intervals. Wounds were identified individually, photographed and mapped on the body of each animal. Individual wounds were scored based on severity according to the following criteria:A pinpoint or pinprick wound, not penetrating dermis, round in shape and no bigger than 5 mm in diameterAny wound not penetrating dermis and >5 mm in length or diameterA wound penetrating dermis, round in shape, no bigger than 5 mm in diameterA wound penetrating dermis and >5 mm in length or diameterTo obtain WSs, animals were sedated with 10–20 mg kgEach quadrant  was assigned a score corresponding to the most severe wound found in that quadrant. All quadrant scores were summed to give a total WS (minimum 0 and maximum 16). Wound scoring was conducted by observers blinded to experimental group assignments. A veterinarian, who was blinded to the conditions of the trial, was responsible for all clinical decisions pertaining to wounds present on individuals. No wounds observed during this study were deemed to require veterinary intervention27 Animals were trained on a pellet delivery apparatus with a computer touch screen and a lighted lever. The monkeys learned to hold the lever, then release and touch a white key, but not a red key, when it appeared on the screen. Probe trials then measured how quickly an animal could adjust its response if the white key turned to red in the time between releasing the lever and touching the screen.The SSRT procedure is considered to measure the ability to inhibit an action . SSRT was tested from 1000 hours to 1100 hours 3 days per week as previously described.31 and was performed as previously described.27 The ID/ED task was conducted at the same time of day for a given animal (1100 hours or 1400 hours) and typically took 4.7 days, on average, to complete. All other behavioral testing was suspended during ID/ED testing. The number of errors during each task and the average time to completion of a trial (latency time) were calculated. This task was performed using a touch-screen computer and consisted of up to eight discrimination-learning stages wherein one of two stimuli, consisting of a background solid shape and foreground line, on the screen is reinforced.32 The ID shift occurs when the shape/line pairs change but the same aspect that was previously rewarded  is still reinforced. The ED shift occurs when the shape/line pairs change again, but the reinforced stimulus is shifted from the background shape to one of the foreground lines. Errors in either stage were summed by counting any incidence of touching the shape not associated with reinforcement. ID/ED errors represent the number of errors, ID and ED stages to reach a preset success criterion .The ID/ED task assesses attentional set shifting27 Briefly, animals were anesthetized and underwent PET scans on a high-resolution research tomography PET scanner. At the beginning of the scan, [11C]raclopride was administered as a bolus and subsequently maintained as continuous infusion. D2/D3 receptor binding potential (BPND) was calculated using a simplified tissue reference model33 for each region of interest identified in the earlier magnetic resonance imaging scans.Parametric images of binding potentials were generated from [11C]raclopride dynamic PET to determine DA D2/D3 receptor binding. Magnetic resonance imaging for regions of interest, including dorsal striatum, caudate nucleus and putamen was performed as previously documented 1 month before the [11C]raclopride scan.n=12) and vehicle-only (n=4)-treated groups, animals initially received oral food vehicle twice daily for 4 weeks. The animals then received twice-daily oral administration (~0800 hours and 1700 hours) of either 5 mg of guanfacine  delivered in the vehicle  or vehicle alone for 4 weeks. Animals were observed an additional 4 weeks after the cessation of therapy with continued twice-daily administration of vehicle. Three animals in the guanfacine-treated group were removed from the study as a result of refusal to accept oral treatment, and their results were discarded. After the end of the initial study design, WSs were collected at 4–6 week intervals until wounding returned to baseline levels.Animals were stratified into four groups according to baseline WSs, and one animal was randomly selected from each severity group to serve as a vehicle-only-treated control. After separation into guanfacine  using mixed model linear regression with repeated measures. Change in WSs from before and after treatment for guanfacine versus vehicle-only-treated controls were compared using a Mann–Whitney test.All statistical analyses were performed using GraphPad Prism 6.0  or Stata , and 16 has been measured using the SSRT procedure in both humans34 and laboratory animals such as rodents and monkeys.35 Each individual's average WS at 7 YoA was compared with SSRT recorded at 2.5 YoA and found to be positively correlated  on the ID and ED shift tasks performed on a touch-screen apparatus by each animal at 2.5, 3, 4 and 4.5 YoA. ID/ED performance did not vary significantly among previous treatments  at any time point (P=0.6293). Comparing subjects' ID/ED errors at all time points to their WS at 7 YoA revealed a significant positive correlation . Based upon this, we compared baseline WS and ID/ED errors at successive ages separately . The number of ID/ED errors at ages 2.5 and 3 had no discernable relationship with wounding . However, at 4 YoA, the correlation between number of ID/ED errors committed and later WS showed a trend toward a positive correlation , with this relationship becoming statistically significant at 4.5 YoA  for three regions of interest known to have high densities of dopaminergic synapses - the dorsal striatum, caudate nucleus, and putamen - were calculated based on [11C]raclopride PET scans of animals performed at 5 YoA. We compared D2/D3 receptor BPND with each individual's average WS representing severity of SIB at 7 YoA (n=13) and found a positive correlation for each of the regions of interest , and an individual with high WS and high D2/D3 BPND  or vehicle only (n=4) by mouth twice daily after 4 weeks of baseline measurements and continuing through week 8. WSs were recorded at 2-week intervals from week 0 through week 12, then at 4–6 week intervals, thereafter until the effects of guanfacine treatment were observed to have waned  tasks was also correlated with increased severity of SIB in our animals. Similarly, degree of cognitive impairment correlates with SIB in human pediatric cases39 and in adult schizophrenia and borderline personality disorder.15 Interestingly, the relationship between this measure of cognitive performance and SIB appeared to develop as our animals matured, trending toward correlation at 4 YoA and becoming significant at 4.5 YoA. Similarly, alterations in impulsivity have been demonstrated to be a significant predictor of SIB in children with autism spectrum disorder.39In our cohort of rhesus macaques, poorer performance on cognitive tasks early in life was associated with increased severity of SIB in adulthood. Impulsivity in these animals, as measured by SSRT at 2.5 YoA, was positively correlated with WS at 7 YoA. This finding mirrors the human condition; impulsive behavior in children is associated with SIB in Cornelia de Lange, Fragile X, Lowe and Prader–Willi syndromes,11C]raclopride at 5 YoA positively correlated with wounding severity at 7 YoA, suggesting that increased D2/D3 receptor availability is correlated with increased wound severity. Increased receptor binding potential could reflect decreases in endogenous ligand. In Parkinson's disease, which is characterized by a decrease in central nervous system DA, one study demonstrated increased D2/D3 receptor binding by [11C]raclopride in affected patients compared with age-matched controls.40 A similar increase in D2/D3 receptor binding and decreased DA has been observed ex vivo in the brains of Lesch–Nyhan syndrome patients.41 Our findings of increased D2/D3 receptor binding therefore suggest that these macaques may have had decreases in intrasynaptic DA or increases in receptor expression at 5 YoA, which were not present at other time points but nevertheless predicted the severity of the SIB at 7 YoA. This mirrors our attentional set shifting results, which also developed a relationship with SIB only as the monkeys aged. Indeed, although [11C]raclopride binding was not directly correlated with ID/ED errors in our animals, decreases in DA function have been correlated with impaired performance on attentional set shifting tasks in humans.42 In addition, research into Parkinson's disease43 and HIV-associated cognitive decline44 suggests that damage to the dopaminergic system may be permanent, which could explain the relationship between temporally distant measures in our monkeys. We currently have only historic cognitive and D2/D3 receptor binding potential data to compare to more contemporary WSs in these animals, but determination of whether changes in these systems and their relationship to SIB severity are permanent represents an important direction for future studies.We also observed that DA D2/D3 receptor binding by [45 It has previously been demonstrated that guanfacine improves impulsivity in rats in a similar manner as dopaminergic agonists,46 and improves cognitive performance on working memory tasks via enhanced function of the prefrontal cortex.47 In addition, guanfacine is known to alter DA signaling48 and decrease the symptoms of Tourette's disorder in children, a disease characterized by decreased DA function in the striatum.49 It thus seems possible that guanfacine decreased SIB severity via temporary correction of the changes we found in the dopaminergic system. We also found that guanfacine had a lasting effect past the cessation of drug administration. The elimination half-life of guanfacine is 13–14 h in children and 17 h in adults, suggesting that its pharmacological effects should not last beyond 2–3 days after cessation of drug administration.50 Despite this, guanfacine-treated animals were not observed to return to similar levels of wounding as vehicle-only-treated controls until 13 weeks after the end of treatment (study week 21). One potential explanation for this latent effect in the face of a short drug half-life is the potential that improvement was not caused by the drug itself, but by a withdrawal effect. However, this would not explain why treatment animals were already trending toward improvement in comparison with vehicle-only-treated controls during treatment weeks 6 and 8. A more likely explanation would be that the correction of DA function led to more persistent downstream effects. With DA's known role in reward-based learning, it could be that there was a temporary correction of whatever aberrant learning process leads the animals to experience SIB as a rewarding event. This ‘relearning' while on treatment could then theoretically outlast the effects of the drug itself. Treatment with extended release naltrexone was shown to have similar positive effects and outlast the duration of the drug treatment in another study of rhesus macaques with SIB.23 Naltrexone's efficacy is presumably owing to disruption of the reinforcing effects of SIB by blockade of the endogenous opioid release accompanying injury, and a similar ‘relearning' process could help explain the effects seen after treatment. This highlights one potential area for future study: the examination of DA and cognitive function both during and after treatment with guanfacine.Guanfacine significantly decreased the wounding associated with SIB in our monkeys, having the greatest effect in those animals with the highest WSs. Although previous case reports have suggested the potential for guanfacine to treat SIB in both children and NHPs, this is the first report of a controlled prospective study demonstrating its efficacy in alleviating SIB. Guanfacine, a preferential α2A agonist, improves neurotransmission by inhibiting postsynaptic signal disruption mediated by cyclic adenosine monophosphate.27 we cannot completely rule out what effect previous psychostimulant drug administration may have had on the development of SIB within this cohort. Further work is needed to compare these SIB macaques with humans and other cohorts of SIB and normal macaques to determine how the pathophysiology compares among these groups. SIB in humans with intellectual deficits has been treated with some success by increasing functional serotonin levels, whether by treatment with selective serotonin reuptake inhibitors51 or by dietary supplementation with serotonin containing foods.52 Similarly, SIB in rhesus macaques has been successfully treated via hyper-supplementation of tryptophan, serotonin's amino-acid precursor28 or by selective serotonin reuptake inhibitors.53 Naltrexone has been a drug of much interest in treating human SIB following Sandman's work linking beta-endorphin cleavage and SIB, although the results in subsequent clinical trials have been mixed.54 Alterations in beta-endorphin metabolism have also been suggested in NHPs with SIB,55 and the recent report of successful treatment of macaque SIB with long-acting naltrexone further supports this hypothesis.23NHPs have not been extensively utilized to model human SIB for a variety of practical and ethical reasons. The prospect of purposefully inducing SIB in NHPs for modeling purposes would be ethically questionable, and assembling a large enough cohort of animals with spontaneously occurring disease is made difficult by the sporadic nature of SIB in macaques, and the fact that it is often not detected until after the age at which animals are typically assigned to other experimental protocols. However, our findings support the notion that the pathophysiology of SIB is similar in humans and NHPs, and an existing cohort such as the one we worked within for this study represents a unique opportunity to explore underlying mechanisms and potential treatments of SIB. Although there was no significant relationship between historical drug therapies from the animals' original study or administration of drugs on behavioral or neuroimaging data,15 We have also shown that treatment with guanfacine, an α2A agonist similar to one previously utilized to treat human SIB, was also effective in treating SIB in our cohort of macaques.24 The increased BPND found in SIB is likely related to the decreased intrasynaptic DA, which in turn may have been ameliorated by treatment with guanfacine. These results hold the promise to help elucidate the problem of SIB not only in captive NHPs but potentially also in their human counterparts as well.The present study adds to and expands the growing body of literature, suggesting that SIB in humans and NHPs may arise from similar mechanisms and thus share potential therapeutic targets. Herein, we have demonstrated that NHPs exhibiting SIB as adults have decreased DA function and display cognitive deficits as juveniles, both of these have also been seen in the context of human SIB.


PER1 and BMAL1) mRNA levels, respectively. The AD patients exhibited significantly longer inactivity interval during the 24 h and significantly higher number of daytime naps than controls. Daily profiles of melatonin levels exhibited circadian rhythms in both groups. Compared with controls, decline in amplitude of the melatonin rhythm in AD patients was not significant, however, in AD patients more melatonin profiles were dampened or had atypical waveforms. The clock genes PER1 and BMAL1 were expressed rhythmically with high amplitudes in both groups and no significant differences in phases between both groups were detected. Our results suggest moderate differences in functional state of the circadian system in patients with mild form of AD compared with healthy controls which are present in conditions of their home dwelling.Alzheimer's disease (AD) is a neurodegenerative disease often accompanied with disruption of sleep-wake cycle. The sleep-wake cycle is controlled by mechanisms involving internal timekeeping (circadian) regulation. The aim of our present pilot study was to assess the circadian system in patients with mild form of AD in their home environment. In the study, 13 elderly AD patients and 13 age-matched healthy control subjects (the patient's spouses) were enrolled. Sleep was recorded for 21 days by sleep diaries in all participants and checked by actigraphy in 4 of the AD patient/control couples. The samples of saliva and buccal mucosa were collected every 4 hours during the same 24 h-interval to detect melatonin and clock gene ( PER1,2, CRY1,2, REV-ERBα, and BMAL1. As a result of the molecular clock mechanism, circadian expression of PER1,2, CRY1,2, and REV-ERBα is in anti-phase to that of BMAL1 (reviewed in  with a constant period of 24 h. The extra sum-of-squares F test was used for comparison, and cosine curve parameters, such as amplitude  were applied. Where appropriate, data were checked for normal distribution.For analysis of the overall rest/activity and sleep/wake daily patterns and of the nighttime and daytime sleep parameters, data from sleep diaries (13 controls and 13 AD patients) and/or from Actiwatch record analyses (4 controls and 4 AD patients) were used.Sleep time during the 24-h period was analyzed in all participants of the study based on records in their sleep diaries. The total time of sleep duration during the 24-h period , expressThe analysis of sleep parameters was performed by the Actiwatch software, and/or using records of the sleep diaries, separately for the night sleep and the daytime naps. The interval designated in the diaries by the subjects as time of falling asleep at night and waking up in the morning was considered the nighttime sleep and the intervals designated by the subjects as short sleep during the day were considered as naps. In one AD patient , the sleep was fragmented, occurring in regular intervals throughout the day and night and, therefore, the nighttime sleep and daytime naps could not be distinguished. The couple of the AD#103 patient/control was thus excluded from this part of analysis. For all other subjects, the nighttime sleep was more or less consistent and clearly distinguishable from the sporadic daytime napping. The actigraphs of 4 control/AD patient couples recorded throughout the 21-day period are depicted in The mean assumed nighttime sleep duration determined from the sleep diaries was 7.3 ± 0.3 h (n = 12) in controls and 9.1 ± 0.7 h (n = 12) in AD patients. For the AD patient/control couples, whose sleep parameters were analyzed by the Actiwatch software, it was 7.8 ± 0.6 h (n = 4) in controls and 9.4 ± 0.4 h (n = 4) in AD patients. Although both measurements showed longer assumed sleep duration in AD patients than in controls, the differences between both groups were not statistically significant, be they detected by the diary records or Actiwatch software . The analysis of the sleep parameters by the Actiwatch software detected the number and duration of wake bouts, percentage of real sleep duration, sleep efficiency and fragmentation index  than in controls (0.25 ± 0.13. n = 12) . The same borderline significance was reached also for data acquired from Actiwatch analysis  where the mean number of daytime naps was significantly higher in AD patients  than in controls . Moreover, the Actiwatch analysis suggested that duration of naps might be longer in AD patients  than in controls , but the difference was not statistically significant .The distribution of individual data for sleep parameters and daytime naps in each of the 4 control/AD patient couples is depicted in In each subject, the melatonin levels were determined at 9 time points during the 24 h. The daily profiles of the mean melatonin levels for the groups of controls and AD patients  are depicted in Examination of individual melatonin profiles revealed a high variability within both groups  but not significant difference between the clock gene expression profiles in AD patients and controls . Moreover, comparison of acrophases between both groups by unpaired Student's t-test did not reveal significant differences .The  rhythms  exhibiten rhythm  was in oIn this pilot field study, we examined actual state of the circadian system in patients with mild form of AD and healthy age-matched controls who lived together in their home dwellings and were thus exposed to the same environmental cues of their everyday life. Our results suggest that under real-life conditions, circadian regulation of behavior, melatonin levels and peripheral clock gene expression in the AD patients may modestly differ compared with the controls. The limitation of this study is a low number of enrollees ; this was mainly caused by the special arrangement of the study. Due to the relatively high inter-individual variability of the data even in control elderly subjects, the low number of participants likely accounts for the fact that our results demonstrate trends rather than significant differences.In AD patients, the circadian activity/rest rhythm was maintained, however, their inactivity interval during the 24 h assessed by sleep diaries was significantly longer compared with that in controls. The analysis of individual sleep/wake cycles revealed that the AD patients and controls exhibited more or less consistent rhythm in nighttime sleep and daytime wakefulness; only in 1 out of 13 patients the rhythm was completely abolished and the sleep occurred in bouts equally distributed over the 24 h period. Subsequent analysis of sleep parameters in 4 AD patient/control couples by Actiwach revealed that whereas the AD patients spent more time inactive in bed, their sleep was likely of poorer quality compared with controls. However, the worse sleep quality in AD patients was only suggested because differences in sleep efficiency and fragmentation index between both groups did not reach statistical significance. This might be related with the low number of subjects which we could include in the actigraphic study due to limited number of the devices. Additionally, the data revealed that the AD patients slept more often during the day as their number of daytime naps was significantly higher than that in controls, be it assessed by sleep diaries in all subjects or by Actiwatch in 4 couples. Analysis of individual data revealed that the napping was present in nearly all AD patients (in 12 out of 13 patients) but less often in controls (in 8 out of 13 controls). Interestingly, one AD patient exhibited a better nighttime sleep quality than all examined controls (sleep efficiency higher than 90% and fragmentation index only about 20%) but the same patient also had the highest count of daytime naps. This case result suggests that the higher number of naps in AD patients might be a consequence of sleep/wake cycle disruption rather than an aftereffect to compensate the poor nighttime sleep quality.Our data demonstrating the longer inactivity during the 24 h in AD patients compared to controls in their home-dwellings are in agreement with previously published findings in hospitalized patients , 45. WitThe mean melatonin profiles exhibited circadian rhythms both in AD patients and controls. Decrease in amplitude of the melatonin rhythm in AD patients compared to controls was not significant, however, inspection of the individual profiles suggested a higher incidence of the rhythm disruptions  in AD patients than in control subjects. Generally high inter-individual variability was found in both groups and, therefore, we cannot be sure that the indicated differences were not due to relatively small sample size employed in the study. Nevertheless, another study confirmed decrease in melatonin rhythm amplitude already in preclinical cognitively intact patients . The stuIn our study, we cannot exclude a possible masking effect of environmental cues, mainly the light, on the endogenous melatonin production, because light was controlled only during the sleep hours on the night when samples were collected. Therefore, although we could not confirm a statistically significant effect of the disease on melatonin levels under real-life conditions, we cannot completely rule out this effect on the endogenous mechanism producing melatonin rhythm. On the other hand, we can exclude the possibility that the differences in melatonin profiles demonstrated in this study in AD patients were due to their different light exposure because the protocol of the study was arranged to ensure that the AD patient/control couples were together throughout the study and were thus exposed to the same environmental cues. In accordance with our results, another hormonal marker controlled by the circadian clock, cortisol, exhibited a significant circadian rhythm in moderately demented home-dwelling AD patients whose activity/rest cycle was disrupted . This reIn summary, our results suggest that compared with their caregivers of the same age and exposed to the same environmental cues, the AD patients exhibit differences in activity/rest cycles which are manifested as significantly higher duration of the inactivity during 24 h and more daytime naps. In AD patients, in spite of the significant disruption in activity/rest and sleep/wake cycles, only marginal disruption, if any, in regulation of melatonin and clock gene expression profiles were detected. Therefore, the endogenous mechanism generating the circadian rhythmic signal is likely preserved during the mild stage of the disease. However, it is necessary to mention that the healthy controls were the patients' spouses who took care of them likely around the clock which might contribute to undervaluation of the results. Nevertheless, we believe that the experimental design of this pilot field study, testing the subjects in real-life situation, has its obvious relevance because it may reveal the effect of AD on adaptability of the circadian system to environmental conditions.S1 TableClinical status of the patients was characterized using Mini-Mental State Examination (MMSE) and Functional Activities Questionnaire (FAQ) , 50. Cha(DOCX)Click here for additional data file.