DNA sequencing of anatomy lab cadavers to provide hands-on precision medicine introduction to medical students.

BACKGROUND: Medical treatment informed by Precision Medicine is becoming a standard practice for many diseases, and patients are curious about the consequences of genomic variants in their genome. However, most medical students' understanding of Precision Medicine derives from classroom lectures. This format does little to foster an understanding for the potential and limitations of Precision Medicine. To close this gap, we implemented a hands-on Precision Medicine training program utilizing exome sequencing to prepare a clinical genetic report of cadavers studied in the anatomy lab. The program reinforces Precision Medicine related learning objectives for the Genetics curriculum. METHODS: Pre-embalmed blood samples and embalmed tissue were obtained from cadavers (donors) used in the anatomy lab. DNA was isolated and sequenced and illustrative genetic reports provided to the students. The reports were used to facilitate discussion with students on the implications of pathogenic genomic variants and the potential correlation of these variants in each "donor" with any anatomical anomalies identified during cadaver dissection. RESULTS: In 75% of cases, analysis of whole exome sequencing data identified a variant associated with increased risk for a disease/abnormal condition noted in the donor's cause of death or in the students' anatomical findings. This provided students with real-world examples of the potential relationship between genomic variants and disease risk. Our students also noted that diseases associated with 92% of the pathogenic variants identified were not related to the anatomical findings, demonstrating the limitations of Precision Medicine. CONCLUSION: With this study, we have established protocols and classroom procedures incorporating hands-on Precision Medicine training in the medical student curriculum and a template for other medical educators interested in enhancing their Precision Medicine training program. The program engaged students in discovering variants that were associated with the pathophysiology of the cadaver they were studying, which led to more exposure and understanding of the potential risks and benefits of genomic medicine.

Morphometric analysis of arteriolar tortuosity in human cerebral white matter of preterm, young, and aged subjects.

Arteriolar tortuousities, consisting of vascular coils, loops, and spirals, appear in white matter in a subset of human cerebral vessels. Computerized morphometry was used to analyze brain sections from a broad age range of subjects to determine whether tortuosity is a phenomenon of aging or is associated with leukoaraiosis (LA) or Alzheimer disease (AD). Autopsy brains were studied from 55 subjects ranging in age from 23 weeks postconception to 102 years. Fourteen aged subjects were diagnosed with LA and 7 with AD. By using computerized morphometry, vascular curl (curvilinear length/straight length) was measured in white matter arterioles in 100-microm-thick, alkaline phosphatase-stained sections. Aging subjects, compared with young subjects, showed significant increases in both the prevalence and severity of tortuosity. Curl scores in aged subjects with LA or AD were not significantly different from aged controls without LA or AD. We conclude that 1) tortuous vessels are extremely rare in preterm babies, children, or young adults; 2) significant tortuosity, as indicated by elevated curl scores, begins in middle age; 3) tortuosity does not appear in a subset of aged individuals regardless of longevity; and 4) tortuosity does not appear in a subset of individuals with either LA or AD.

Vascular dementia in leukoaraiosis may be a consequence of capillary loss not only in the lesions, but in normal-appearing white matter and cortex as well.

We investigated capillary density in 12 subjects with leukoaraiosis (LA), in 9 age-matched normal subjects, in 7 cases of Alzheimer's disease (AD), and 4 after whole-brain irradiation for brain tumors. In the LA study (which as been published), autopsy brains were evaluated by MRI. The presence of LA was indicated by confluent or patchy areas of hyperintensity in the deep white matter. We employed a stereology method using computerized image processing and analysis to determine microvascular density. Afferent vessels (arterioles and capillaries, but not veins or venules) were stained for alkaline phosphatase in 100 microm thick celloidin sections. Microvascular density in LA lesions in the deep white matter (2.56%) was significantly lower than in the corresponding deep white matter of normal subjects (3.20%, p=0.0180). LA subjects demonstrated decreased vascular density at early ages (55-65 years) when compared to normal subjects. Our findings indicate that LA affects the brain globally, with capillary loss, although the parenchymal damage is found primarily in the deep white matter. In ongoing studies of the deep white matter in AD brains, we found a pattern of decreased vascular density compared to normal, as well as an age-related decline. In the four irradiated brains, we found very low vessel densities, similar to those found in LA, without an additional age-related decline.

Germinal matrix cells associate with veins and a glial scaffold in the human fetal brain.

Germinal matrix (GM) in the subventricular zone (SVZ) includes progenitor cells of neurons and glia, which migrate from the SVZ to regions where they become integrated into the developing brain. In the human fetal brain, GM cells pack into high density clusters that encircle GM veins producing a profile we describe as a venous cuff. Venous cuffs are, in turn, encircled by GFAP-positive astrocytes that project processes through the cuff to the venous wall. The high cell density exhibited by cuffs, as well as their association with astrocytes, are reminiscent of features associated with chain migration. However, chain migration has not been associated previously with veins. We suggest that the GM cuff cells may represent a distinct subset of GM cells that migrate away from the GM on a pathway consisting of a vein and its associated astrocytic scaffold.

Temporal expression pattern of cerebrovascular endothelial cell alkaline phosphatase during human gestation.

In premature human neonates, immaturity of cerebral vessels can contribute to clinical problems such as germinal matrix hemorrhage and white matter damage. Afferent cerebral vessels in the brain of term babies express alkaline phosphatase (AP), an ectoenzyme located on the surface of endothelial cells. Using AP enzyme histochemistry we have examined the cerebrovasculature of premature live-born human neonates to determine when cerebral afferent vessels begin to express AP. Brains were collected at autopsy and processed for histological examination. AP-stained vessel density in the periventricular white matter was quantified using digital imaging and automated morphometry. Babies born prior to 28 wk gestation display few AP-positive vessels in the periventricular white matter, whereas, babies born after 28 wk gestation exhibit an AP-positive vascular pattern that resembles the adult pattern. In contrast, immunostaining for collagen revealed an extensive vascular network in both early and late gestation infants. Our measurements indicate that neonates born prior to 28 wk gestation are characterized by immature cerebral white matter afferent vessels and raise the possibility that the immaturity compromises vascular function.

Increase of white matter string vessels in Alzheimer's disease.

String vessels are collagenous structures connected to capillaries. They have no endothelial cells or lumen. We assessed collagen IV-labeled string vessels in the white matter (WM) of subjects with Alzheimer's disease (AD) (n = 12) and non-AD controls (n = 11) using 100 microm celloidin sections. Ten standard fields were digitally captured and the number and length of normal vessels and string vessels were quantified by computerized image analysis. The WM of the AD-diagnosed individuals contained more strings per mm2 (3.95 +/- 0.49) than comparable WM from controls (1.36 +/- 0.39) (p = 0.0005) and had increased total string vessel length in mm/mm2 (AD = 0.29 +/- 0.04; control = 0.10 +/- 0.03; p = 0.0015). There was a 25% increase (not statistically significant) in vessel density in mm/mm2 in AD subjects (AD = 11.88 +/- 0.87; control = 9.53 +/- 0.78; p = 0.06), presumably due to brain atrophy in the white matter. Although vessel length was slightly increased in AD subjects, they still had more than double the string length per total vessel length (AD = 2.88 +/- 0.38) compared to controls (1.36 +/- 0.27) (p = 0.0057). This increase in string vessels in the white matter of AD subjects suggests a decrease in vascular supply in this disease.

The Role of Extracellular Ca2+ in the Activation of Pectinaria Oocytes.

Several events are associated with fertilization in oocytes. Two such events are an increase in cytoplasmic Ca2+ concentration and the resumption of meiosis. Oocytes of the marine annelid, Pectinaria gouldii, are in metaphase I arrest when they are spawned. In this report we investigate the relationship between Ca2+ and resumption of meiosis in this species. Meiosis in unfertilized oocytes could be re-initiated with the divalent cation ionophore, A23187. Oocytes in Ca2+ free sea water, however, did not resume meiosis in the presence of the ionophore. Furthermore, it was observed that Ca2+ must be present for at least 15 min following ionophore treatment for meiosis to resume. These results suggest that extracellular Ca2+ is required for the re-initiation of meiosis in this species.

Apoptosis in leukoaraiosis lesions.

Leukoaraiosis (LA), an age-related degenerative condition, appears as an area of hyperintense signal in the deep white matter on MRI. It may be caused by chronic ischemia. LA lesions are characterized by demyelination, loss of glial cells, spongy appearance, and occlusion of veins and venules by collagenous thickening of the vessel walls. Since necrosis is not obvious in LA lesions, we investigated the occurrence of apoptosis. We obtained 1.5-cm-thick coronal brain slices at autopsy from two patients with LA. MRI was performed on the brain slices. Blocks were fixed in formalin, embedded in paraffin, and sectioned. Sections were stained by several methods including the TUNEL method for DNA fragmentation. Some TUNEL-positive cells showed nuclear morphology indicative of apoptosis. In case 1, TUNEL-positive cells were more numerous in an LA lesion than in nearby unaffected white matter (P=0.008). In case 2, LA lesions were examined in six areas; left and right frontal, middle, and occipital slices. TUNEL-positive cells were more numerous in the LA lesions than in nearby white matter (P=0.002). We also found TUNEL-positive cells in the cortex and in the walls of blood vessels. In case 1, more severe venous collagenosis was found in the LA lesion, which was near the cortex, than in the periventricular area, where venous collagenosis and LA are more commonly found. The presence of numerous scattered cells in the LA lesions showing DNA fragmentation suggests that those cells are damaged and dying, at least some by apoptosis. The apoptosis in the white matter adjacent to the LA lesions suggests progressive cell loss and expansion of the LA lesions.

Venous collagenosis and arteriolar tortuosity in leukoaraiosis.

Leukoaraiosis (LA), an age-related white matter degeneration, is thought to be caused by chronic ischemia. To understand the pathogenesis of LA, we studied the pathology, particularly of the blood vessels, in 186 brains [84 of them with magnetic resonance imaging (MRI)] over the past 10 years. With normal aging, there is gradual thickening of the walls of periventricular veins and venules with collagen subtypes I and III. This venous collagenosis (VC) was increased in brains with LA. Occasionally, LA lesions are not periventricular, but nearer the cortex. In such cases, the most severe VC occurs in the LA lesion rather than near the ventricle. Therefore, LA and VC are not independent degenerative processes coincidentally found near the ventricles, and although damage to the ependyma could be a cause of VC, it cannot be the only cause. Whether VC precedes LA is unknown, but our experience suggests that severe VC is usually accompanied by LA. Arteriolar tortuosity, another age-related vascular pathology, is common in LA. Our thick celloidin sections show three-dimensional views of tortuous arterioles. The tortuosity is much more severe in the white matter and there is considerable loss of parenchyma around them. Staining for collagen IV in the basal lamina reveals tortuous vessels in an "empty bag" that represents the limits of the surrounding parenchyma. These enlarged perivascular spaces correspond to état criblé. The demyelination in LA lesions is accompanied by loss of cells, mostly oligodendrocytes. In studies of apoptosis in LA, we found increased apoptosis within the lesion compared to the surrounding white matter. In conclusion, our studies support the concept that LA results from chronic ischemia due to age-related vascular pathology.

Subependymal veins in premature neonates: implications for hemorrhage.

The germinal matrix contains a concentrated network of blood vessels. The unusual structural qualities of these vessels are implicated as a factor underlying the high incidence of hemorrhage that occurs in the germinal matrix of prematurely born neonates. The present study is a histologic analysis of an postmortem examination series of brains collected from neonates born between 23 weeks gestation and term and is designed to determine if subependymal veins can be recognized in neonates born at the limits of viability, approximately 23 weeks gestation. Alkaline phosphatase histochemistry is used to differentiate cerebral afferent from efferent vessels. The results demonstrate that precursors of the subependymal veins can be recognized as early as the twenty-third gestational week. These veins increase progressively in diameter from 23 weeks to term, but the wall of the veins, which at early stages consists of endothelial cells only, does not thicken until after postconception week 36. Thus in all premature neonates, including the youngest capable of independent existence, the subependymal veins are present and appear vulnerable to rupture. These data support our suggestion that the structural immaturity of these veins in premature neonates is causally related to the high incidence of germinal matrix hemorrhage in these patients.

Microvascular changes in the white mater in dementia.

Our studies of the brain microvascular system have focused on some aspects not commonly studied by other research groups because we use some techniques not often used by others. Our observations tend to add new details to the pathological picture rather than contradict the mainstream findings. We use large, thick celloidin sections which provide a three dimensional view of vascular networks, and alkaline phosphatase (AP) staining which allows one to differentiate between afferent and efferent vessels. We found millions of lipid microemboli in the brains of patients after cardiac surgery, and concluded that they caused vascular dementia in many patients. We previously proposed an animal model of vascular dementia using brain irradiation, which induces capillary loss. Lipid emboli might also be used to create an animal model of vascular dementia. The deep white matter is vulnerable to chronic hypoperfusion because the blood vessels supplying this region arise from the border-zone and have the longest course of all vessels penetrating the cerebrum. In cases with leukoaraiosis (LA), we found periventricular venous collagenosis (PVC), resulting in stenosis. Thirteen of 20 subjects older than 60 years had PVC, and 10 of 13 subjects with severe PVC had LA. Vascular stenosis might induce chronic ischemia and/or edema in the deep white matter, leading to LA. We suggest three mechanisms for a possible genetic predisposition to PVC: i) a predisposition to excessive venous collagenosis; ii) an indirect effect that causes chronic periventricular ischemia with a reactive over-production of collagen; and iii) mechanical damage to small vessels due to increased pulsatile motion. We found tortuous arterioles supplying the deep white matter beginning at about age 50. We also found a trend toward an increase in tortuosity in LA. If tortuosity is a factor in LA, it is probably significant in only a subset of cases. String vessels, remnants of capillaries, occur commonly in the brain, and are increased in ischemia, AD, and irradiation. Capillary injury or shutdown of blood flow can lead to capillary loss and string vessel formation. We found string vessels in brains from preterm babies to the very old. They seem to disappear after some months or years. We found an early loss of capillaries in LA, followed in a few years by the disappearance of string vessels. LA lesions do not progress to cortical cavitating lesions. Our findings raise three questions. 1. Why is the capillary loss arrested before infarction? 2. Why is there a floor below which the vascular density will not fall? 3. Why does the process which initiates string vessels shut down? We explain the vascular changes in LA as follows. LA induces apoptosis with loss of oligodendrocytes. Capillaries and neuropil are lost. Increased oxygen extraction from the blood in the deep white matter in LA implies that there are too many cells for the remaining capillaries. Thus, the capillaries appear to die first. But why do they stop dying? Perhaps a minimum number of capillaries are needed to transport the arterial blood to the venous system. Once the capillaries stop dying, no more string vessels are formed, and the string vessels gradually disappear.

Immunolocalization of tight junction proteins in blood vessels in human germinal matrix and cortex.

Brain development occurs in a specialized environment maintained by a blood-brain barrier (BBB). An important structural element of the BBB is the endothelial tight junction (TJ). TJs are present during the embryonic period, but BBB impermeability accrues over an extended gestational interval. In studies of human premature infants, we used immunomicroscopy to determine if amounts of the TJ proteins ZO-1, claudin and occludin increase with gestational age in vessels of germinal matrix (GM) and cortex. By 24 weeks postconception (PC), TJ proteins were present in both GM and cortical vessels, but immunoreactivity in the GM of the youngest subjects was less than in older subjects. At 24 weeks PC, TJ protein immunoreactivity in GM vessels was less than in cortical vessels suggesting that TJ maturation progresses along a superficial to deep brain axis. This concept correlates with conclusions from previous analyses of the expression of brain endothelial cell alkaline phosphatase (AP) activity. AP appears in cortical vessels before appearing in deep white matter and GM vessels. Together, these data indicate that differentiation of some functional specializations is still in progress in GM vessels during the third trimester. This maturation could relate to the pathogenesis of germinal matrix hemorrhage-intraventricular hemorrhage.

Morphometric assessment of collagen accumulation in germinal matrix vessels of premature human neonates.

Germinal matrix haemorrhage in premature neonates is commonly attributed to vascular immaturity, possibly related to an abbreviated process of angiogenesis. Terminal steps in the progression of angiogenesis are the formation of a subendothelial basal lamina containing collagen IV and an extracellular matrix containing collagens I and III. Immature vessels would predictably be deficient in these collagen subtypes. We analysed germinal matrix (GM), cortical, and white matter (WM) vessels with antibodies specific for collagens I, III and IV to test the hypothesis that GM vessels are immature. Brains were collected during post-mortem from prematurely born human neonates ranging in age from 17 weeks to 36 weeks postconception. All GM vessels were immunoreactive for collagen subtypes I, III and IV. Using digital image analysis, collagen IV immunoperoxidase-labelling was measured in vessels in GM, cortex and WM. Intensity values in GM and WM were normalized relative to cortical intensity within the same subject. At week 17 of gestation, GM vessels exhibited a higher concentration of collagen IV than did WM or cortical vessels. Regression analysis demonstrated that collagen intensity in GM was greater than that in cortex and WM at all stages. We conclude that GM vessels in even the youngest, prematurely born, viable neonates do not exhibit evidence of structural immaturity. The high incidence of GM haemorrhage in premature neonates may be related to factors other than a deficiency in accumulated collagen.

Formation of sea urchin primary mesenchyme: cell shape changes are independent of epithelial detachment.

Primary mesenchyme cells in sea urchin embryos emerge from the epithelium of the blastula wall and move into the blastocoel in a process referred to as ingression. Ingressing cells are bottle-shaped with their basal end protruding into the blastocoel and their apical end narrowed into a thin strand. Soon after becoming bottle-shaped, the ingressing cells move into the blastocoel and form the primary mesenchyme which ultimately assembles the skeleton of the larva. Primary mesenchyme formation requires zygotic gene expression but the pathways that lead from gene expression to ingression are not known. In this study cordycepin, an inhibitor of mRNA adenylation, and thus translation, is used to determine when ingression becomes sensitive to disruption of gene expression. When added to embryos 4 h before the onset of ingression, cordycepin inhibits the release of mesenchyme cells from the epithelium. Interestingly, though, it does not inhibit the shape changes that mark the onset of ingression. If added 2 h before the onset of ingression, cordycepin does not interfere with the formation of primary mesenchyme. These results indicate that both cell shape changes and the detachment process require zygotic transcripts and that the shape changes can occur independent of detachment.

A morphological analysis of the first cleavage mitotic cytoskeleton isolated from sea urchin embryos (Strongylocentrotus droebachiensis).

Morphological changes in the mitotic cytoskeleton (MC) that occurred through the course of the first cleavage of Strongylocentrotus droebachiensis, cultured at 8°C or at 0°C, temperatures within the natural range for this species, have been investigated. Electron microscopy of MCs isolated from zygotes has revealed that they consisted largely of microtubules (mts). Thus, the morphology of these MCs is derived from the arrangement of the mts which form them. During anaphase, astral rays elongated while kinetochore fibers shortened. Asters enlarged during anaphase as a result of two events: astral ray lengthening and centrosphere enlargement. At the end of anaphase, asters of 8°C MCs filled the entire cell volume. The pattern of changes that occurred in 8°C mitotic apparatuses (MAs) also occurred in 0°C MCs. The observation of asters in 0°C MCs is contradictory to that of Stephens ('72b), who reported that 0°C MCs in this species were anastral. However, in 0°C metaphase MCs, the astral rays and spindle fibers were not as long as those in 8°C MCs. Also in 0°C MCs, the centrosphere was largely filled with dense material, whereas the centrosphere in 8°C MCs was larger and contained little dense material. Asters of 0°C MCs did not attain a large enough size to fill the egg volume completely, as did asters of 8°C MCs.

Immunohistochemical localization of a tenascin-like extracellular matrix protein in sea urchin embryos.

We have used polyclonal antisera raised against vertebrate tenascin to identify and localize tenascin-like proteins in the developing sea urchin. These antisera recognize high-molecular weight proteins on immunoblots of sea urchin embryo homogenates that are similar in size and appearance to tenascin from vertebrates. These proteins appear as a doublet with an apparent molecular weight of 150 kDa and a larger, broad band with an apparent molecular weight of 350 kDa. Whole mounts of sea urchin embryos and larvae were stained with one of these antisera. The anti-tenascin stained the surface of primary mesenchyme cells during their phase of active migration. This staining was sensitive to detergent, suggesting that the protein recognized by the antiserum was associated with the cell surface. During later stages of development, the bulk of the antitenascin staining was found dispersed throughout the blastocoel matrix, and was no longer sensitive to detergent. We conclude that sea urchins express tenascin-like proteins during early stages of development, and that these proteins may play a role associated with primary mesenchyme cell morphogenesis.

Quantification of afferent vessels shows reduced brain vascular density in subjects with leukoaraiosis.

PURPOSE: To investigate vessel density changes with increasing age in three areas of the brain and to correlate these changes with leukoaraiosis (LA) on the basis of magnetic resonance (MR) images and location in deep white matter (WM). MATERIALS AND METHODS: Internal review board approval or informed consent from next of kin was not required. Brains of 21 subjects (mean age, 72.5 years; 12 men, nine women) were evaluated at autopsy with MR imaging. The presence of LA was indicated by confluent or patchy areas of hyperintensity in deep WM. Microvascular density (percentage of vessel area divided by total area) in subjects with LA was measured with computerized morphometric analysis in LA lesions, healthy-appearing WM at MR imaging, and the cortex. These measurements were compared with each other and with measurements from corresponding areas in healthy subjects. Afferent vasculature was stained with alkaline phosphatase in celloidin sections. Hypotheses were tested with computation of a series of repeated-measures linear mixed models. RESULTS: Autopsy brains from 12 subjects with LA (mean age, 72 years; six men, six women) and nine subjects without LA (mean age, 73 years; six men, three women) were studied. Afferent microvascular density +/- standard deviation in LA lesions in deep WM (2.56% +/- 1.56) was significantly lower than that in corresponding deep WM of healthy subjects (3.20% +/- 1.82) (P = .018). Subjects with LA demonstrated decreased afferent vascular density at early ages in all three areas of the brain when compared with healthy subjects of the same age. CONCLUSION: Findings of decreased afferent vascular density in the area of LA and outside the lesion indicate that LA is a generalized cerebrovascular disease process rather than one confined to deep WM.

Maternal stores of α subtype histone mRNAs are not required for normal early development of sea urchin embryos.

The ability to specifically delete the store of maternal α-subtype histone mRNAs stored in the egg pronucleus has allowed us to examine the role of this major fraction of the maternal mRNA in the early development of the sea urchinStrongylocentrotus purpuratus. The egg nucleus was removed by centrifugation, and the resulting enucleate half eggs were fertilized. These haploid andromerogones lacked any stored α-subtype histone mRNAs. However, when grown in parallel with control embryos, they showed identical cleavage cycles, cell numbers, and patterns of cell differentiation. Measurements of the amount of α-histone mRNA in these andromerogones showed that there was no premature synthesis of α-histone mRNAs to compensate for the deleted maternal pool. Instead embryonic synthesis was normal in timing of initiation and duration. the ability of these embryos to develop into highly differentiated larvae without their maternal α-subtype histone mRNA pool suggests that this pool is not a critical component of early development per se. This suggestion is strengthened by the observation that the primitive sea urchinEucidaris tribuloides naturally lacks this maternal histone mRNA store. Evolutionary implications are discussed.

Anatomical analysis of the developing cerebral vasculature in premature neonates: absence of precapillary arteriole-to-venous shunts.

Germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH) commonly affects premature neonates. The gravity of the consequences associated with GMH-IVH makes it a major concern in their care. GMH-IVH begins in the germinal matrix tissue and is, most commonly, the result of venous rupture. Arteriole-to-venous precapillary shunts in the cerebrum of premature neonates could, if present, lead to elevated venous pressure in the germinal matrix and, thus, would be an important etiological factor. We report an autopsy study, involving 33 cases of premature neonates, designed to determine whether precapillary arteriole-to-venous shunts are present in the cerebral vasculature. Brain tissue was embedded in celloidin, sectioned into 100- micro m-thick slices and stained using alkaline phosphatase enzyme histochemistry, a method that distinguishes afferent from efferent vessels. Our sections, which are ideal for tracing vessels over long distances and for displaying patterns of branching and connections with other vessels, indicate that precapillary arteriole-to-venous shunts are not a major influence on cerebral blood flow in babies born at 23 wk gestation or later. The cerebral vasculature in one baby, who died at 24 wk postconception, included shunt-like connections, whereas in 34 babies shunts were not identified. We conclude that precapillary arteriole-to-venous shunts are not a significant factor leading to GMH.