Winter in the Forest, Dalecarlia

Illustration by Anshelm Schultzberg courtesy of the John Herron Art Museum

Effects of Peroral Omega-3 Fatty Acid Supplementation on Cerebrospinal Fluid Biomarkers in Patients with Alzheimer’s Disease: A Randomized Controlled Trial—The OmegAD Study

Background: Studies have suggested a connection between a decrease in the levels of polyunsaturated fatty acids (PUFAs) and Alzheimer’s disease (AD). We aimed to assess the effect of supplementation with omega-3 fatty acids (n-3 FAs) on biomarkers analyzed in the cerebrospinal fluid (CSF) of patients diagnosed with AD. / Objective: To investigate the effects of daily supplementation with 2.3 g of PUFAs in AD patients on the biomarkers in CSF described below. We also explored the possible correlation between these biomarkers and the performance in the cognitive test Mini-Mental State Examination (MMSE). / Methods: Thirty-three patients diagnosed with AD were randomized to either treatment with a daily intake of 2.3 g of n-3 FAs (n  =  18) or placebo (n  =  15). CSF samples were collected at baseline and after six months of treatment, and the following biomarkers were analyzed: Aβ 38, Aβ 40, Aβ 42, t-tau, p-tau, neurofilament light (NfL), chitinase-3-like protein 1 (YKL-40), acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), soluble IL-1 receptor type II (sIL-1RII), and IL-6. / Results: There were no significant differences between the groups concerning the level of the different biomarkers in the CSF at baseline. Within the treatment group, there was a small but significant increase in both YKL-40 (p = 0.04) and NfL (p = 0.03), while the other CSF biomarkers remained stable. / Conclusion: Supplementation with n-3 FAs had a statistically significant effect on NfL and YKL-40, resulting in an increase of both biomarkers, indicating a possible increase of inflammatory response and axonal damage. This increase in biomarkers did not correlate with MMSE score. / Trial registration: clinicaltrial.gov Identifier: NCT00211159

The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease

Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson’s disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson’s disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson’s disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells

Intravenous Inoculation of a Bat-Associated Rabies Virus Causes Lethal Encephalopathy in Mice through Invasion of the Brain via Neurosecretory Hypothalamic Fibers

The majority of rabies virus (RV) infections are caused by bites or scratches from rabid carnivores or bats. Usually, RV utilizes the retrograde transport within the neuronal network to spread from the infection site to the central nervous system (CNS) where it replicates in neuronal somata and infects other neurons via trans-synaptic spread. We speculate that in addition to the neuronal transport of the virus, hematogenous spread from the site of infection directly to the brain after accidental spill over into the vascular system might represent an alternative way for RV to invade the CNS. So far, it is unknown whether hematogenous spread has any relevance in RV pathogenesis. To determine whether certain RV variants might have the capacity to invade the CNS from the periphery via hematogenous spread, we infected mice either intramuscularly (i.m.) or intravenously (i.v.) with the dog-associated RV DOG4 or the silver-haired bat-associated RV SB. In addition to monitoring the progression of clinical signs of rabies we used immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to follow the spread of the virus from the infection site to the brain. In contrast to i.m. infection where both variants caused a lethal encephalopathy, only i.v. infection with SB resulted in the development of a lethal infection. While qRT-PCR did not reveal major differences in virus loads in spinal cord or brain at different times after i.m. or i.v. infection of SB, immunohistochemical analysis showed that only i.v. administered SB directly infected the forebrain. The earliest affected regions were those hypothalamic nuclei, which are connected by neurosecretory fibers to the circumventricular organs neurohypophysis and median eminence. Our data suggest that hematogenous spread of SB can lead to a fatal encephalopathy through direct retrograde invasion of the CNS at the neurovascular interface of the hypothalamus-hypophysis system. This alternative mode of virus spread has implications for the post exposure prophylaxis of rabies, particularly with silver-haired bat-associated RV

Murine prenatal expression of cholecystokinin in neural crest, enteric neurons, and enteroendocrine cells

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5–9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development. Dev Dyn 1999;216:190–200 . © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35165/1/9_ftp.pd

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered

Plasma gut hormone levels in 37 patients with pheochromocytomas

Pheochromocytomas are usually recognized by the effects of overproduction of catecholamines, but there are clinical features that cannot be ascribed to catecholamine excess that may be due to vasoactive peptides. We, therefore, measured blood levels of vasoactive intestinal peptides (VIP), substance P, somatostatin (SS), and motilin in 50 instances in 37 patients with pheochromocytomas-21 malignant, 10 benign intra-adrenal, and 6 ectopic (5 paracardial and 1 perirenal). Hormone levels were considered raised if the level was more than 3 S.D. above the mean value found in 52 healthy subjects. Of the 37 patients, 20 (54%) had an abnormality in 1 or more gut hormone levels. The most common abnormality was a raised SS in 9/37 (24%). In addition to these, however, 3 (8%) others had raised VIP, 5 (13.5%) raised motilin, and 3 (8%) raised substance P. Patients with benign adrenal adenomas had raised levels of SS and substance P. Ectopic pheochromocytomas produced only SS in addition to catecholamines, but malignant pheochromocytomas could secrete all 4 peptides, and more than 1 in the same patient. We conclude that pheochromocytomas may secrete multiple vasoactive peptides, and they are more likely to do so if malignant. Somatostatin is the most commonly secreted peptide and is found with benign adrenal and ectopic (paracardiac) tumors. If the level of more than 1 peptide is elevated, the likelihood of malignancy is significantly increased . Les phéochromocytomes sont généralement déceléspar les effets dûs à la surproduction de catécholamines, mais certains troubles ne peuvent être attribués à ce phénomène et relèvent peut être de l'action de peptides vasoactifs. Les auteurs se sont donc attachés à doser dans le sang le VIP, la substance P, la somatostatine (SS), et la motiline. Ces dosages furent pratiqués 50 fois chez 37 malades porteurs de phéochromocytomes: 21 malins, 10 bénins et 6 ectopiques (5 paracardiaque et 1 péri-rénal). Les taux des hormones furent considérés comme élevés lorsque leur niveau fut supérieur à plus de 3 fois le taux de 52 sujets sains. Sur les 37 malades 20 (54%) présentaient un excès d'une ou de plusieurs hormones digestives. L'anomalie constatée la plus fréquente fut l'élévation de la SS (9 fois sur 37 soit 24%). Ajoutée à ce fait fut l'élévation de la VIP chez 3 sujets (8%), de la motiline chez 5 (13.5%) et de la substance P chez 3 (8%). Les phéochromocytomes bénins surrénaliens présentaient à la fois une élévation du taux de la SS et de la substance P. Les phéochromocytomes ectopiques en revanche présentaient seulement une élévation de la SS. Les phéochromocytomes malins pouvaient sécréter les 4 peptides ou plus d'un chez le même malade. En conclusion les phéochromocytomes peuvent secréter de multiples peptides vasoactifs et plus particulièrement lorsqu'ils sont malins. La SS est la substance qui est la plus souvent secrétée et elle est trouvée dans les tumeurs bénignes surrénaliennes ou ectopiques. Si plus d'une de ces substances est produite en excès les risques de malignité de la tumeur sont significativement plus importants. Los feocromocitomas generalmente son diagnosticados por los efectos del exceso de producción de catecolaminas pero hay características clínicas que no pueden ser atribuidas al exceso de catecolaminas y que pueden ser más bien manifestación de péptidos vasoactivos. Hemos establecido los niveles sanguíneos del péptido intestinal vasoactivo (VIP), de la sustancia P, de la somatostatina (SS), y de la motilina en 50 determinaciones en 37 pacientes con feocromocitomas; 21 malignos, 10 benignos intra-adrenales, y 6 ectópicos (5 paracardiales y 1 perirrenal). Se consideró que los niveles hormonales estaban elevados cuando el nivel era de más de 3 de desviación estandar sobre el valor promedio en 52 individuos normales. De 37 pacientes, 20 (54%) presentaron un valor anormal en 1 o más determinaciones del nivel de hormonas intestinales. La anormalidad más común fue la elevación de la SS en 9/37 (24%). Además de esto, sinembargo, otros 3 (8%) presentaban elevación de VIP, 5 (13.5%) elevación de sustancia P. Los adenomas suprarrenales benignos exhibieron niveles elevados de SS y de sustancia P. Los feocromocitomas ectópicos demostraron producción sólo de SS además de catecolaminas, pero los feocromocitomas malignos demostraron ser capaces de secretar todos los 4 péptidos, y más de 1 en el mismo paciente. Hemos llegado a la conclusión de que los feocromocitomas pueden secretar múltiples peptidos vasoactivos y que ésto tiende a ocurrir cuando son malignos. La SS es el péptido más frecuentemente secretado y se lo encuentra en los tumores suprarrenales benigno y ectópico (paracardiacos). Si se encuentran niveles elevados de más de 1 péptido, la posibilidad de malignidad aparece significativamente aumentada.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41274/1/268_2005_Article_BF01655534.pd

Causal Inference in Observational Studies and Experiments: Theory and Applications

This thesis consists of six papers that study the design of observational studies and experiments. Paper I proposes strategies to consistently estimate the average treatment effect of the treated using information derived from a large number of pre-treatment measurements of the outcome. The key to this strategy is to use two-level time-series model estimates to summarize the inter-unit heterogeneity in the sample. It is illustrated how this approach is in line with the conventional identifying assumptions, and how sensitivity analyses of several key assumptions can be performed. Paper II contains an empirical application of the identification strategy proposed in Paper I. This study provides the first causal analysis of the demand response effects of a billing demand charge involuntarily introduced to small and medium sized electricity users. Paper III proposes strategies for rerandomization. First, we propose a two-stage allocation sample scheme for randomization inference to the units in balanced experiments that guarantees that the difference-in-means estimator is an unbiased estimator of the sample average treatment effect for any experiment, conserves the exactness of randomization inference, and halves the time consumption of the rerandomization design. Second, we propose a rank-based covariate-balance measure which can take into account the estimated relative weight of each covariate. Paper IV discusses the concept of optimal rerandomization. It is shown that depending on whether inference is to be drawn to the units of the sample or the population, the notion of optimal differs. We show that it is often advisable to aim for a design that is optimal for inference to the units of the sample, as such a design is often near-optimal also for inference to the units of the population. Paper V summarizes the current knowledge on asymptotic inference for rerandomization designs and proposes some simplifications for practical applications. Drawing on previous work, we show that the non-normal sampling distribution of the difference-in-means test statistic approaches normal as the rerandomization criterion approaches zero. Furthermore, the difference between the correct non-normal distribution and the proposed approximation based on a normal distribution is in many situations negligible even for near optimal rerandomization criteria. Paper VI investigates and clarifies the relation between the traditional blocked designs and rerandomization. We show that blocking and rerandomization is very similar, and in some special cases identical. Moreover, it is shown that combining blocking and rerandomization is always at least as efficient as using only rerandomization, but the difference is in many cases small

Causal effects in mediation analysiswith limited-dependent variables

Mediation is used to separate direct and indirect effects of an exposure variable on anoutcome variable. In this thesis, a mediation model is extended to account for censoredmediator and outcome variable. The two-part framework is used to account for thecensoring. The counterfactual based causal effects of this model are derived. A MonteCarlo study is performed to evaluate the behaviour of the causal effects accounting forcensoring, together with a comparison with methods for estimating the causal effectswithout accounting for censoring. The results of the Monte Carlo study show that theeffects accounting for censoring have substantially smaller bias when censoring is present.The proposed effects also seem to have a low cost with unbiased estimates for samplesizes as small as 100 for the two-part mediator model. In the case of limited mediatorand outcome, sample sizes larger than 300 is required for reliable improvements. A smallsensitivity analysis stresses the need of further development of the two-part models