Variability of the midfacial muscles: analysis of 50 hemifacial cadaver dissections.

The region of the midface represents a challenging area to both reconstructive and aesthetic surgeons. An anatomic study was performed that attempted to identify patterns and variations of the muscular anatomy. The goals of this study were twofold: to identify patterns and variability of the midfacial muscles that might impact on reconstructive efforts and to attempt to correlate this anatomy with features of the overlying soft tissues, specifically the nasolabial crease. Fifty hemifacial cadaver dissections were performed. The information collected was assembled into a large data base, and statistical significance was analyzed using Fisher's exact probability test. Results demonstrated that, although a great degree of variability exists with respect to the midfacial muscles, seven distinct patterns of these muscles did emerge. The most common pattern was the presence of a levator alae nasi, levator labii superioris, and zygomaticus major, which occurred in 44 percent of specimens. Specimens that possessed a risorius, zygomaticus minor, or both, were relatively uncommon. The consistent presence of the levators suggests adding a superior vector to recreate a smile in facial reanimation surgery. Two important anatomic variations were noted. A bifid zygomaticus major was found to be present in 34 percent of individuals. Because the inferior bundle had a dermocutaneous insertion, this anomaly may represent the anatomic correlate of a cheek "dimple." A second anomaly noted was the lateral cheek crease, which appeared to be associated with a cutaneous attachment from the underlying platysma muscle. However, no correlation could be found for facial muscle pattern and the overlying nasolabial crease structure. This lack of correlation may indicate that the facial muscles alone do not dictate the structure of the nasolabial crease and that other dynamic factors are involved in determining this feature of the aging face.

Double or bifid zygomaticus major muscle: anatomy, incidence, and clinical correlation.

The anatomy of the double or bifid zygomaticus major muscle is investigated in a series of 50 hemifacial cadaver dissections. The double zygomaticus major muscle represents an anatomical variation of this muscle of facial expression. This bifid muscle originates as a single structure from the zygomatic bone. As it travels anteriorly, it then divides at the sub-zygomatic hollow into superior and inferior muscle bundles. The superior bundle inserts at the usual position above the comer of the mouth. The inferior bundle inserts into the modiolus below the corner of the mouth. The incidence of the double zygomaticus major muscle was 34% in the present study, as it was found to be present in 17 of 50 cadaver dissections. This study shows that variation in the individual morphology of the mimetic muscles can be a common finding. Clinically, the double or bifid zygomaticus major muscle may explain the formation of cheek "dimples." The inferior bundle was observed in several specimens to have a dermal attachment along its mid-portion, which tethers the overlying skin. When an individual with this anatomy smiles, traction on the skin may create a dimple due to this dermal tethering effect.

Anatomy of a "black eye": a newly described fascial system of the lower eyelid.

The anatomy of a black eye is examined in a series of cadaver dissections in which a previously unreported fascial system of the lower eyelid is identified. This fascia originates at the orbital rim, and is in continuity with the orbital septum and with the periosteum of the orbital floor and anterior maxillary wall. This fascia contributes to the thickened area along the orbital rim called the arcus marginale. At the level of the orbicularis oculi muscle, this fascia was noted microscopically to fuse with a fibrous septa of the superficial cheek fat. This creates one long continuous membrane from the orbital rim above to the cheek skin below. Dye injection techniques show that this membrane is impermeable and traps injected dye in the same place where a black eye forms. After periorbital injury, extravasated hemoglobin pigment is confined to the area above the cutaneous insertion of this membrane. This fascial system has been named the septum malaris: malar describes its origin along the orbital rim of the cheek, and septum further describes the partitioning nature of this ultra-thin membrane.

Terminal nerve distribution to the urethra and bladder neck: considerations in the management of stress urinary incontinence.

PURPOSE: Recent reports have suggested an increased incidence of intrinsic sphincter dysfunction, most of which seems to appear following the failure of a previous, usually vaginal, surgical repair. Our studies attempt to define more precisely the neuroanatomical relationships that exist in the region of the bladder neck and proximal urethra, and between the urethra and anterior vaginal wall. MATERIALS AND METHODS: We dissected the pelves of adult female cadavers and step sectioned them at 4 mm. intervals. Several staining methods were used on each section to identify and document the position of the nerves and vascular structures between the vaginal wall and urethra. RESULTS: A rich plexus of blood vessels and nerves with ganglia is located between the vaginal wall, and the proximal urethra and bladder neck. The greatest concentrations of nerves are in the 4 o'clock and 8 o'clock positions but nerve fibers are identified throughout the loose areolar tissue planes through which vaginal surgery for stress urinary incontinence is often performed. CONCLUSIONS: When performing surgical procedures for the correction of stress urinary incontinence, the possibility that denervation and devascularization of the terminal urethra and bladder neck secondary to surgical dissection could contribute to the subsequent development of intrinsic sphincter dysfunction should be considered.

A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide.

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.

YY1 and Sp1 transcription factors bind the human transferrin gene in an age-related manner.

The iron-binding protein transferrin has major roles in transporting, delivering, and sequestering ferric ions acquired by body tissues. Yet, during aging, serum transferrin levels decrease in humans. Likewise, in transgenic mice carrying chimeric human transferrin transgenes, liver expression of transferrin transgenes decreases with age. The aging regulation is due to decreased gene transcription. Electrophoretic mobility shift assays and antibody-recognition have revealed the binding of 5' regulatory elements of the human transferrin gene by three YY1 proteins, called YY1, YY1-a, and YY1-b, and an Sp1-a transcription factor. An age-related increase in YY1-a and YY1-b binding activities and a decrease in Sp1-like binding activity were shown. Since Sp1 is a positive transcription factor and YY1 can be a negative transcription factor, the alterations in their binding with age could cause the decreased transcription of the human transferrin transgene, and also the age-related decreased serum transferrin levels in humans.

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver.

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice.

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.

Monocytes become macrophages; they do not become microglia: a light and electron microscopic autoradiographic study using 125-iododeoxyuridine.

This light and electron microscopic autoradiographic study of stab injuries in the spinal cord of mice evaluated the ultrastructural characteristics of cells labeled by incorporation of the thymidine analogue 125I-5-iodo-2'-deoxyuridine (I-UdR), injected one day prior to injury. I-UdR was used instead of tritiated thymidine (H-TdR) because H-TdR can be reutilized and is therefore not a suitable pulse label for long-term studies of cell migration. Using serial thick and thin sections for autoradiography 614 labeled cells were identified. Labeled cells included 545 monocytes/macrophages, 50 lymphocytes, 17 pericytes, one endothelial cell, and one arachnoid cell. No labeled cell had the morphology of microglia. We concluded that macrophages in stab injuries of the spinal cord of mice are derived from blood monocytes. Blood-derived lymphocytes are also involved in the reaction to spinal cord stab injury. Microglia are not blood-derived and are not seen as a transitional form in the differentiation of monocytes to macrophages.

Evidence for turnover of DNA in the nucleus and in the cytoplasm of adult post-mitotic neurons in vivo.

Experiments were designed to determine if turnover of DNA occurs in post-mitotic neurons of adult mammals. Three-month-old laboratory mice, Mus musculus, and white footed mice, Peromyscus leucopus, were given three injections of 3H-thymidine (3H-TdR, 16 muCi/g body weight) at noon, 4 pm and 8 pm, and were killed serially beginning 1 h after the last injection. The brain stem was removed from the animals and autoradiographed. Some of the sections were treated with DNase prior to autoradiography. A sensitive and accurate method of autoradiographic grain count analysis was used to measure the grain counts over areas of neuron nuclei, neuron cytoplasm and background areas of the slide (Cameron, Pool and Hoage, 1979). There was a significant elevation of grains per unit area above background over both the nucleus and the cytoplasm in animals killed 24 h after the last 3H-TdR injection. Counts were reduced to background level by prior DNase treatment. This shows that the grains over the nucleus and the cytoplasm were due to label in the DNA. Changes in the grain counts with time were subjected to least squares regression analysis. In the mouse both the nucleus and the cytoplasmic grain counts showed a decreasing slope which was significantly different from a slope of zero when the data were fitted to either a linear model or a log-linear model. The data showed a somewhat better fit to the log-linear model. The labelled nuclear DNA in the mouse had a calculated half-life of about 502 h while the labelled cytoplasmic DNA had a calculated half-life of about 97 h. Our ability to measure and to characterize DNA turnover in specific in vivo cell types allows us to test theories about its functional role.

Non-specific esterase activity in reactive cells in injured nervous tissue labeled with 3H-thymidine or 125iododeoxyuridine injected before injury.

Tritiated thymidine (3H-TdR) injected before a stab wound of the spinal cord or transection of the hypoglossal nerve has resulted in many labeled reactive cells in the CNS after injury, most of which have the ultrastructural features of microglia. To test for the possible origin of these labeled cells from monocytes, we examined them for the presence of sodium fluoride- (NaF) sensitive non-specific esterase (NSE), an enzyme characteristic of monocytes. Some of the labeled cells in stab wounds had NaF-sensitive NSE, but no such cells were found in the nucleus of the injured hypoglossal nerve. To test for the possibility that the NSE-negative labeled cells had been labeled by reutilization of 3H-TdR, we used 125I-5-iodo-2'-deoxyuridine (125I-UdR), a thymidine analogue with a much lower rate of reutilization, to label blood mononuclear cells prior to either a spinal cord stab wound or hypoglossal axotomy. The number of labeled cells was decreased in the spinal cord wound, but more than half were NSE-negative. No labeled blood mononuclear cells were found in the hypoglossal nucleus, although there was no decrease in the hyperplasia of unlabeled non-neuronal cells. When 125I-UdR was injected on the fourth day after hypoglossal axotomy, or when both 3H-TdR and 125I-UdR were injected simultaneously before hypoglossal axotomy, many labeled cells were found in the hypoglossal nucleus, indicating that 125I-UdR can be used by the reactive cells and that it did not inhibit their proliferation. Therefore, the microglial cells that proliferate in response to peripheral nerve injury are not recently derived from any type of circulating large blood mononuclear cell. The most likely explanation for the presence of the 3H-TdR-labeled cells in the nucleus of the injured hypoglossal nerve is that they were proliferating intrinsic cells labeled by reutilization of 3H-TdR.

Effects of intestinally absorbed thymidine on tritiated thymidine utilization.

Experimental evidence presented suggests that [3H]TdR can be rapidly and efficiently transported from the intestine to the systemic circulation. This pathway for thymidine transport may be physiologically important since administration of cold thymidine in the drinking water enhances the utilization of a parenterally injected dose of [3H]TdR in several body tissues of the mouse.

Unstable nuclear DNA in hypoglossal neurons of adult mice.

To demonstrate the existence of unstable or metabolic DNA in normal mammalian neurons and to study the effect of peripheral nerve injury on this metabolic DNA, adult mice were given repeated injections of high doses of 3H-thymidine (3H-T) on the day before injury to the left hypoglossal nerve. The animals were killed at intervals up to 33 days after the injections of 3H-T. Analyses of grain counts showed a low but significant elevation in the number of radioautographic grains per unit area of hypoglossal neuronal nuclei above background levels for up to 5 days after 3H-T injection. Digestion of the tissue with DNase lowered the nuclear grain counts to background levels, confirming that the DNA was indeed labelled. Although there was a loss of labelled material from the neuronal nuclei with time, there was no difference between injured and uninjured neurons at any of the intervals tested after injection of 3H-T.

Fine structure of reactive cells in injured nervous tissue labeled with 3H-thymidine injected before injury.

To examine the fine structure of blood mononuclear cells in injured nervous tissue, mice were given repeated injections of 3H-thymidine with the last injection at least 16 hours before injury. Under ether anesthesia the animals either were given a stab wound to the spinal cord or had their left hypoglossal nerve transected. The animals were killed at 2, 4, 8, or 16 days after injury. Tissue sections containing the spinal cord wound or both hypoglossal nuclei were prepared for electron microscopic radioautography, and all labeled cells were photographed. About half the labeled cells in the injured spinal cords and almost all the labeled cells in the nuclei of the injured hypoglossal nerves had nuclei with dark staining peripheral heterochromatin, dark cytoplasm with long cisternae of granular endoplasmic reticulum, and other ultrastructural features characteristic of the cells usually identified as microglia. The remaining labeled cells in the injured spinal cords were macrophages, fibroblasts, cells with pale nuclei, some of which contained cytoplasmic filaments, and vascular cells. Since uninjured nervous tissue has extremely few labeled cells and since 3H-thymidine should be available for only a short time following injection, most of the labeled cells in this experiment should be derived from blood mononuclear cells. However, the possibility is discussed that some or all of the labeled cells may be intrinsic cells proliferating in response to the injury and labeled through reutilization of labeled DNA precursor material.

The use of elemental iodine to enhance staining of thin sections to be viewed in the electron microscope.

A noticeable increase in contrast is observed when thin sections, stained with Reynolds lead citrate, are subsequently exposed to elemental iodine vapor for 30 seconds. There is no loss of ultrastructural detail, and there is no evidence of harmful iodine contamination of the microscope after prolonged study of such material. It is recommended that this simple procedure be used when other methods of staining have not proved adequate.