By F. Mine-Boss. Trinity International University. 2019.
Here cheap keppra 500 mg fast delivery, we detail the placental explant culture method employed in our labo- ratory to collect extracellular vesicles which are known to be released by the human placenta throughout pregnancy from 6 weeks of gestation discount 250mg keppra overnight delivery. Using this method purchase generic keppra line, at least three different populations of placental extracellular vesicles can be simultaneously collected from each placental sample buy keppra 500 mg fast delivery, allowing for comparative analysis of the cargos and downstream effects of the different types of extracellular vesicles produced by the human placenta. Key words Vesicle, Trophoblastic debris, Microparticle, Explant culture, Placenta 1 Introduction In addition to the secretion of hormones and other soluble factors, the production of extracellular vesicles by the human placenta has recently been recognized as a novel mode of feto-maternal com- munication that is important for both physiological adaptations during normal human pregnancy [1–4] and the pathophysiology of obstetric diseases such as preeclampsia [5–8]. The effects of placental extracellular vesicles on recipient cells are likely to be mediated by their protein, lipid, and nucleic acid cargos. As the outermost surface of the human placenta is covered by the multinucleated syncytiotrophoblast, a large range of extracellular vesicles can be produced by the human placenta, ranging in size from macro-vesicles (20–150 μm), to microvesicles (100–1000 nm), to exosomes and other nano-vesicles (20–100 nm) [10, 11]. While placental extracellular vesicles have been detected in the blood of pregnant women from as early as 6 weeks of gestation, their levels in the circulation are much lower than that of maternal platelet- derived and endothelial cell-derived extracellular vesicles . Chamley Therefore, it has been challenging to isolate circulating placental extracellular vesicles for downstream analysis. This is compounded by a lack of robust placenta-specifc markers that can be used for the purifcation of placenta-derived extracellular vesicles from the blood [13, 14]. Therefore, in order to characterize placental extra- cellular vesicles to better understand their potential functions and to identify novel markers for these extracellular vesicles, most current studies have isolated extracellular vesicles from human placentae ex vivo. In the literature, placental macro- and nano-vesicles have predominately been collected by culturing villous placental explants in a static and minimally disruptive system for 24–96 h and isolating the extracellular vesicles by differential centrifugation. In con- trast, three methods have been commonly reported for the collec- tion of placental microvesicles: (1) mechanical dissection/ disruption, (2) placental explant culture, and (3) placental perfu- sion. Depending on the method used to collect placental microves- icles, their cargo and downstream effects can be drastically different [15–17], and it is now established that mechanical disruption of placental villi is a poor method for collecting physiologically relevant microvesicles . For the collection of extracellular vesicles from intact term pla- centae, both placental explant culture and placental perfusion methods can be used, while only the placental explant culture method can be used to isolate extracellular vesicles from frst tri- mester placentae as these placentae are often damaged and lack the depth of villous tissue required to perform perfusion. Chapter 14 has detailed the principles and methods of placental perfusion; thus, this chapter will describe the placental explant culture method in detail and how this can be employed to isolate different size frac- tions of extracellular vesicles simultaneously from the same placen- tal sample by sequential centrifugation. Finally, the characterization of the total protein content as well as the shape and size of extracel- lular vesicles by electron microscopy and nanoparticle tracking analysis, respectively, will be described. Plastic inserts with a 400 μm mesh: Sterilize between use by leaving in 1% bleach for 1 h, leaving in disinfectant (see Note 2) for 72 h, and storing in 70% ethanol at room temperature until required. For mid−/late-gestation placentae, dissect and discard the top 2 mm of the maternal aspect of the placenta, which contains maternal decidual tissue, and dissect out approximately 2cm3 of the underlying villous placental tissue. To increase the rep- resentativeness of sampling, usually at least three areas of the mid−/late-gestation placenta are sampled ranging from the center of the placenta to the periphery, resulting in at least 6cm3 of placental villous tissue. After suffcient washing, further dissect the villous placental tissue into explants of approximately 400 mg (see Note 7). Four placental explants usually generate suffcient extracellular vesicles for physical characterization and protein collection. By this time, the inserts should have dried and can be placed in a 12-well culture plate, creating two compartments (Fig. When adding such reagents, take care to avoid overly diluting the base medium, and if using human serum, as a general rule, this should make Isolation and Characterization of Placental Extracellular Vesicles 121 Fig. In our work, we have frequently cultured placental explants at ambient oxygen levels for 16 h, but culture condi- tions can be easily manipulated in this system (see Note 9). We have also previously reported that culture oxygen conditions (2, 8 and 20%) did not signifcantly affect the number and size of micro- and nano-vesicles extruded from frst trimester human placentae . After 16 h of culture, lift the inserts, each containing a placen- Centrifugation tal explant, out from the wells of the 12-well plate, taking care to decant as much of the culture medium from around the placental explant as possible back into the well. Mix the culture medium in each well by pipetting, and collect the culture medium from all placental explants (in the four culture wells) into one sterile tube. Centrifuge at 2000 × g for 5 min at 4 °C to sediment the pla- cental macro-vesicles and other contaminating cells (red and white blood cells) from the culture medium (Fig. Carefully decant the supernatant resulting from this centrifugation step into a sterile polycarbonate ultracentrifugation tube (see Note 10), and store at 4 °C for up to 48 h prior to ultracentrifuga- tion to isolate the micro- and nano-vesicles. After decanting, resuspend the pellet, containing placental macro-vesicles and contaminating red and white blood cells, in the remaining ~200 μL of supernatant by gently tapping the base of the tube. Remove contaminating red blood cells by adding in 9 mL sterile water and inverting to create a hypotonic environment. This time, the pellet should look white as most red blood cells should be lysed (see Note 12). Insert the tube into a suitable magnet, which traps the Dynabeads against the wall of the tube, and after 10 s, transfer the supernatant containing placental macro-vesicles into a sterile 1. Centrifuge tubes at 8000 × g for 5 min at 4 °C, and after removal of the supernatant by pipetting, the pellet contains the placental macro-vesicle fraction which should be resus- pended in the relevant buffer or media. While the placental macro-vesicle fraction is being purifed, placental microvesicles can also be simultaneously isolated from the supernatant collected in Step 3. To isolate placental microvesicles, the supernatant collected in Step 3 is centri- fuged at 20,000 × g for 1 h at 4 °C (Fig. The resulting pellet containing the placental microvesicle frac- tion should be kept in the fridge, while placental nano-vesicles are isolated from the supernatant. The supernatant from the 20,000 × g centrifugation step (Step 12) should be decanted into a new sterile polycarbonate tube that is rated for ultracen- trifugation at 100,000 × g and centrifuged at 100,000 × g for Isolation and Characterization of Placental Extracellular Vesicles 123 1 h at 4 °C to collect the placental nano-vesicle fraction (Fig. For all vesicle pellets, supernatant that was not completely decanted is removed using a pipette after resting the tubes upright for 5 min at room temperature. Depending on the downstream assay, different solutions can be used to resuspend the vesicle pellets. Resuspend pellets of extracellular vesicles in ultrapure water by Microscopy repeat pipetting with a 1 mL and 200 μL micropipette at least 20 times each (see Notes 15–17). Gently overlay a formvar-coated copper mesh grid onto this droplet to coat the surface for 2 min at room temperature. Carefully wick off excess solution with hardened ashless flter paper, and transfer the copper grid onto a droplet of 2% uranyl acetate for 2 min at room temperature. Carefully wick off excess solution with hardened ashless flter paper, and transfer the copper grid onto a droplet of ultrapure water for 2 min. Repeat Step 5 to remove excess stain, and after wicking off excess solution with hardened ashless flter paper, allow grids to dry at room temperature under a lamp. Sample-coated copper grids are stored sample side up at room temperature and viewed by transmission electron microscopy within 2 h. Turn on the NanoSight system and fush 10 mL of ultrapure water through the tubing and gasket. Connect the syringe to the NanoSight system inlet, and rap- idly load 500 μL of the sample into the NanoSight system (see Note 19). Vesicles should now be apparent on the NanoSight computer, and the focus can be adjusted on the NanoSight machine such that the majority of vesicles have sharp boundaries and perhaps 124 Mancy Tong and Lawrence W. The sample is now ready to be analyzed and the temperature of the stage should be set, usually at 25 °C. In our work, we have typically taken three 30 s recordings of each sample vol- ume, and this is automatically controlled by running a script (Table 1). An example of a script that can be used for analyzing fowing vesicles is provided in Table 1. After the frst set of recordings and analysis, advance the sam- ple by 100 μL in the syringe and do another set of recordings. To obtain representative counts, at the end of this recording, we advanced the sample and counted it three more times, resulting in a total of fve sets of readings (15 recordings of 30 s in total). The average vesicle concentration, mean, and modal size of each set of readings were recorded, and from this, the fnal average concentration, mean, and modal size of all fve sets of readings can be calculated (see Note 21). Taking into account the dilutions performed, the total num- ber of extracellular vesicles in the samples can be calculated, and we typically normalize this value to the weight of the orig- inal placental explants or the protein content of the placental explants (see Note 22). In some studies, fetal bovine serum is frst diluted 1:1 in fresh media and ultracentrifuged up to 120,000 × g for 18 h to remove endogenous extracellular vesicles before being used to supplement culture media [18, 19]. However, recent studies have shown that culture with media supplemented with extra- cellular vesicle-depleted fetal bovine serum reduced cell prolif- eration compared to culture with traditional media [20, 21]. Explants from later gestation placentae can be cut into four smaller pieces to further open up the structure to allow the release of extruded extracellular vesicles into the culture medium. Micropore tape is used to seal the system so that the plate and inserts stay together, but oxygen can still freely fow through into the culture system. In the current literature, the concentration of oxygen used for placental explant culture varies from 2 to 20% oxygen.
Te membranous laby- the basal turn of the cochlea near the round window in the rinth contains endolymph and the sensory epithelium critical scala tympani order 500 mg keppra overnight delivery, the cochlear aqueduct allows communication to its function generic keppra 500 mg overnight delivery. Vestibule Internal Auditory Canal Te vestibule is an ovoid structure centrally located within the labyrinth purchase cheap keppra on line. It is located medial to the middle ear generic 250mg keppra free shipping, posterior Te internal auditory canal transmits the facial and vestibu- to the cochlea, and anterior the semicircular canals. Te lateral wall of the vestibule contains the oval window, which labyrinthine artery, a branch of the anterior inferior cerebellar abuts the middle ear space and is flled by the stapes footplate artery, also courses through the internal auditory canal. Scala tympani Organ of Corti Scala media Reissner’s membrane Scala vestibuli Figure 4-8 Cochlea and cochlear nerve. A cross section through the canal near Te facial nerve leaves the brain stem at the pontomedul- the fundus demonstrates four distinct nerves within the lary junction, where it exits just anterior to the vestibuloco- lumen. Te cisternal segment of the nerve is 15 mm in nerve is anterior and inferior, and the superior and inferior length and travels within the cerebellopontine cistern to the vestibular nerves are posteriorly situated within the canal. Te vertical crest, canal at the porus acusticus, and the intracanalicular segment or Bill’s bar separates the anterior from posterior contents, of the facial nerve is 12 mm in length. Te nerve travels and the transverse crest separates the superior from inferior within the anterior superior portion of the internal auditory contents. Te nerve exits the internal auditory canal at the meatal foramen and becomes the labyrinthine Facial Nerve and Fallopian Canal facial nerve. Te meatal foramen is the narrowest portion of the fallopian canal with an average diameter of 0. At the geniculate ganglion, the nerve ponents of the organs of hearing and balance. Te facial nerve makes its frst turn, or genu, and becomes the tympanic facial runs within the fallopian canal, which is the longest osseous nerve. With this frst genu, the nerve makes a sharp turn from channel for any nerve in the body. Te facial nerve can be the anterosuperiorly directed labyrinthine segment to the subdivided into multiple segments: cisternal, intracanalicular, posteroinferiorly oriented tympanic segment. When 11-mm tympanic segment of the facial nerve travels within undertaking surgery of the temporal bone and lateral skull the tympanic cavity of the middle ear and passes posterior base, surgeons must have a detailed understanding of the superior to the oval window. Te nerve courses superior to facial nerve’s course in order to avoid the devastating com- the cochleariform process along the inferior surface of the plication of facial nerve paralysis. At the inferior margin of the lateral Te facial nerve has a motor and sensory component, and semicircular canal, it makes another turn (the second genu) the sensory component is derived from the nervus interme- and becomes the inferiorly directed vertical segment. Te nerve provides motor innervation vertical segment travels 10 to 14 mm, as the longest segment to the mimetic musculature of the face, posterior belly of the of the intratemporal facial nerve, and exits the temporal bone digastric, stylohyoid muscle, stapedius muscle, and auricular at the stylomastoid foramen. Te nerve provides sensory input including taste Within the temporal bone, there are three branches of sensation to the anterior two thirds of the tongue and somatic the facial nerve. Te next intratemporal branch of the chapter, there is a vast wealth of knowledge worth additional facial nerve is the nerve to the stapedius muscle within study for those interested. Te last intratemporal branch is the the temporal bone, detailed anatomic knowledge is required chorda tympani nerve. Tis nerve arises from the distal to avoid potentially devastating complications such as facial portion of the vertical facial nerve, courses superiorly and paralysis, hearing loss, and vertigo. Te nerve pro- via the study of anatomic treatises as well as cadaveric dis- vides taste sensation to the lateral two thirds of the tongue section prior to tackling surgical procedures in this region. Te size of the middle ear and the mastoid air and clinical evaluation, Plast Reconstr Surg 7. Tis knowledge guides the surgeon in the planning of surgical approaches and Te malar fat pad is a triangular structure superfcial to the in reconstruction of traumatic or surgical defects. It is oriented with its base along the nasolabial fold facilitates the visceral functions, including breathing, smell- and its apex at the zygomatic prominence. In this discussion, the descends and loses volume with age, resulting in the descent anatomy of the region is addressed in a manner similar to a of the facial soft tissues associated with aging. Te zygomatic and buccal branches of the facial nerve lie superfcial to the buccal process, with the The Skin parotid duct running within it. Care should be taken when excising a portion of the buccal fat pad for cosmetic purposes Te skin is a complex organ, composed of superfcial epider- or when using a pedicled buccal fat fap for oral-antral fstula mis and underlying dermis, that provides sensation and pro- closure. Te anatomic and physiologic properties of the skin Multiple other distinct fat pads have been described and play an important role in the fnal esthetics of any facial surgi- should be taken into account, particularly when fat is injected cal procedure (Figure 5-1). Burget and Menick2,3 The Superfcial Musculoaponeurotic System expounded upon this concept. For the muscles of facial expression, similar to the superfcial example, the skin of the nasal dorsum is nearly 3. Tis should be taken into consid- (temporoparietal) fascia superiorly and with the superfcial eration when planning skin resurfacing procedures that rely cervical fascia of the platysma inferiorly. Te superfcial vascular plexus lies between the papillary and reticular dermal layers. Te musculocutaneous arteries provide blood supply deep to the subcutaneous tissue. Six major muscle groups in the head assist with visceral func- tions: orbital muscles, masticatory muscles, muscles of facial expression, tongue muscles, pharynx muscles, and larynx mus- The Facial Layers of the Face: Regional cles. Tis chapter focuses on the masticatory muscles, mimetic Considerations muscles (muscles of facial expression), and tongue muscles. In the upper face, when elevating a coronal or forehead fap, Masticatory Muscles dissection in the avascular plane between the periosteum and temporoparietal fascia protects the temporal branch of the Te muscles of mastication are derived from the frst bran- facial nerve. Nasal subunits: 2A, Tip; 2B, Columellar; 2C, Dorsal; 2D, Right and left dorsal side wall; 2E, Right and left alar base; 2F, Right and left alar side wall. Periorbital subunits: 3A, Lower eyelid; 3B, Upper eyelid; 3C, Lateral canthal; 3D, Medial canthal. Auricular subunits: 8A, Helical; 8B, Antihelical; 8C, Triangular fossa; 8D, Conchal; 8E, Lobe. Table 5-2 Layers of Facial Musculature Some have suggested that there are four layers of facial muscles; layer four is unique because these muscles are inner- Layer 1 Depressor anguli oris, zygomaticus minor, orbicularis vated from their superfcial surface, whereas the muscles in oculi layers one through three receive innervation from their deep Layer 2 Depressor labii inferioris, risorius, platysma, zygomaticus surfaces (Table 5-2). Te regional position of the facial nerve is shown in relation to surgically important anatomic layers. Tese muscles function formation and bolus control by pressing the cheek against the to close the oropharyngeal opening via contraction of the teeth during mastication. Defcit of the hypoglossal nerve results in a protruded tongue pointing toward the injury or lesion. Tis is due to Musculature of the Tongue the fan-shaped insertion of the bilateral genioglossus muscles Te musculature of the tongue is composed of four intrinsic that cross the midline anteriorly. Te intrinsic muscles of the tongue are the superior and inferior longitudinal muscles, the transverse muscles, and the The Facial Nerve vertical muscles. Te extrinsic muscles are the genioglossus, hyoglos- face varies markedly from person to person. All are inner- understanding of the anatomic literature and recognition of Temporalis muscle Insertion on both sides of coronoid process of mandible: Anterior Posterior Superior lateral pterygoid muscle Figure 5-4 Muscles of mastication. Te pterygomandibular ligament A space between the mandible and the medial pterygoid is where local anes- Pterygomandibular thetic is injected for a block of the space inferior alveolar nerve. Tere are commonly anastomoses between the buccal foramen between 6 and 8 mm medial to the tympanomastoid and zygomatic branches, but the temporal and mandibular suture and just lateral to the styloid process. Because bution and branching pattern of the facial nerve become of the degree of morbidity associated with damage to these quite variable. Te a description of the most commonly accepted pattern, repre- temporal branch leaves the parotid and runs within the senting around 24% of individuals. Te fve arch between 8 and 35 cm anterior to the external auditory 15 branches, or rami, of the facial nerve are the temporal (or canal. Te marginal mandibular nerve exits the 16 21 posterior to the superfcial temporal vessels. Dingman and Grabb, in their rior branch is 2 cm posterior to the anterior extent of the classic dissection study, identifed the majority of marginal 17 zygomatic arch. Anterior to the facial vessels, temporalis fascia, or within the temporal fat pad between the the nerve is above the mandibular border 100% of the time. Te number of branches varies from one to four, but two Several landmarks are available in the temporal region. Estimation of the temporal branch distribution can be made Te marginal mandibular nerve is protected throughout by drawing a triangle from the earlobe to the lateral brow the majority of its course by the platysma muscle. About 2 cm 18 and lateral extent of the highest forehead crease or from a lateral to the corner of the mouth, the nerve becomes more point 0. Te distribution of the marginal mandibular nerve Another approach, although somewhat more technique- must be discussed with regard to surgical approaches to the sensitive, is to refect the platysma superiorly and visually lower face, because injury to this nerve results in paralysis of identify and protect the marginal branch of the facial nerve the lip and chin, producing a notable deformity; in addition, (Figure 5-7).
The anesthesia provider must be certain that the scavenging system is operational and adjusted properly to ensure adequate 1720 scavenging purchase keppra 500mg otc. An “active system” uses a central evacuation (vacuum) system to remove waste gases purchase keppra overnight delivery. The “weight” or pressure of the waste gas itself produces flow through a “passive system discount 250mg keppra overnight delivery. Gas-Collecting Assembly The gas-collecting assembly captures excess anesthetic gas and delivers it to the transfer tubing discount keppra amex. Gas passing through these valves accumulates in the gas-collecting assembly and is directed to the transfer means. If this occurs, waste anesthetic gases may overflow the system via the positive-pressure relief valve (closed systems) or through the atmospheric vents (open systems) into the operating room atmosphere. Transfer Means The transfer means carries excess gas from the gas-collecting assembly to the scavenging interface. Some manufacturers color code the transfer tubing with yellow bands to distinguish it from 22-mm diameter breathing system tubing. The two tubes usually merge into a single hose before they enter the scavenging interface. Occlusion of the transfer means can be particularly problematic since it is upstream from the pressure-buffering features of the scavenging interface. If the transfer means is occluded, baseline breathing circuit pressure will increase and barotrauma can occur. Scavenging Interface The scavenging interface is the most important component of the system because it protects the breathing circuit or ventilator from excessive positive or negative pressures. The interface should limit the pressures immediately downstream from the gas collecting assembly to between −0. Positive-pressure relief is mandatory,2 irrespective of the type of disposal system used, to vent excess gas in case of occlusion downstream from the interface. If the disposal system is an “active system,” negative-pressure relief is necessary to protect the breathing circuit or ventilator from excessive subatmospheric pressure. A reservoir is highly desirable with active systems, since it stores waste gases until the evacuation system can remove them. Interfaces can be open or closed, depending on the method used to provide positive- and negative-pressure relief. Open Interfaces An open interface contains no valves and is open to the atmosphere, allowing 1722 both positive- and negative-pressure relief. Open interfaces should be used only with active disposal systems that use a central evacuation system. Open interfaces require a reservoir because waste gases are intermittently discharged in surges, whereas flow from the evacuation system is continuous. Many contemporary anesthesia machines are equipped with open interfaces like those in Figures 25-50A and B. The canister volume should be large enough to accommodate a variety of waste gas flow rates. Gas enters the system at the top of the canister and travels through a narrow inner tube to the canister base. Positive- and negative-pressure relief is provided by holes in the top of the canister. The open interface shown in Figure 25-50A differs somewhat from the one shown in Figure 25-50B. The operator can regulate the vacuum by adjusting the vacuum control valve shown in Figure 25-50B. The vacuum flow rate per minute must equal or exceed the volume of excess gases to prevent spillage. The volume of the reservoir and the flow characteristics within the interface are important. Spillage will occur if the volume of a single exhaled breath exceeds the capacity of the reservoir. The flow characteristics of the system are important because gas leakage can occur long before the volume of waste gas equals the reservoir volume if significant turbulence occurs within the interface. All 1723 closed interfaces must have a positive-pressure relief valve to vent excess system pressure if obstruction occurs downstream from the interface. A negative-pressure relief valve is mandatory to protect the breathing system from subatmospheric pressure if an active disposal system is used. One has positive-pressure relief only; the other has both positive- and negative-pressure relief. Transfer of the waste gas from the interface to the disposal system relies on the “weight” or pressure of the waste gas itself since a negative-pressure evacuation system is not used. The positive-pressure relief valve opens at a preset value such as 5 cm water if an obstruction between the interface and the disposal system occurs. This interface has a positive- pressure relief valve, and at least one negative-pressure relief valve, in addition to a reservoir bag. Figure 25- 51 (right) is a schematic of Dräger Medical’s closed interface for suction systems. A variable volume of waste gas intermittently enters the interface through the waste gas inlets. The reservoir intermittently accumulates excess gas until the evacuation system eliminates it. The operator should adjust the vacuum control valve so that the reservoir bag is properly inflated (A), not over-distended (B), or completely deflated (C). Gas is vented to the atmosphere through the positive-pressure relief valve if the system pressure exceeds +5 cm water. Room air is entrained through the negative-pressure relief valve if the system pressure is more negative than −0. The effectiveness of a closed system in preventing spillage depends on the rate of waste gas inflow, the evacuation flow rate, and the size of the reservoir. Leakage of waste gases into the atmosphere occurs only when the reservoir bag becomes fully inflated and the pressure increases sufficiently to open the positive-pressure relief valve. In contrast, the effectiveness of an 1725 open system to prevent spillage depends not only on the volume of the reservoir but also on the flow characteristics within the interface. Gas-Disposal Assembly Conduit The gas-disposal assembly conduit, or disposal assembly tubing (Fig. It should be collapse-proof and should run overhead, if possible, to minimize the chances of accidental occlusion. Gas-Disposal Assembly The gas-disposal assembly ultimately eliminates excess waste gas (Fig. The most common method of gas disposal is the active assembly, which uses a central evacuation system. A vacuum pump serves as the mechanical flow-inducing device that removes the waste gases usually to the outside of the building. An interface with a negative-pressure relief valve is mandatory because the pressure within the system is negative. A reservoir is very desirable, and the larger the reservoir, the lower the suction flow rate needed. Instead, the “weight” or pressure from the heavier-than-air anesthetic gases produces flow through the system. Positive-pressure relief is mandatory, but negative-pressure relief and a reservoir are unnecessary. Some include venting through the wall, ceiling, floor, or to the room exhaust grill of a nonrecirculating air conditioning system. Hazards Scavenging systems minimize operating room atmosphere contamination, yet they add complexity to the anesthesia system. A scavenging system functionally extends the anesthesia circuit all the way from the anesthesia machine to the ultimate disposal site. Obstruction of scavenging pathways can cause excessive positive pressure in the breathing circuit, and barotrauma can occur. Excessive vacuum applied to a scavenging system can result in undesirable negative pressures within the breathing system. For most contemporary anesthesia workstations, preuse checkout of the scavenging system is a function that must be performed manually by the operator according to the manufacturer’s instructions. Since some anesthesia machines are designed such that ventilator drive gas (oxygen) is also scavenged, the environment in these machine rooms into which the scavenged gas is vented may become highly enriched with oxygen. These sites may contain equipment or materials such as petroleum distillates (pumps/oil/grease) that in the presence of an oxygen- enriched atmosphere could be excessively combustible and present a severe fire hazard.
Probe Selection Probe selection and labeling have direct impact on hybridization assay efﬁciency discount keppra 250mg amex. The ideal probe is single-stranded discount keppra 500 mg without prescription, lacks secondary structure order discount keppra on-line, and does not self anneal buy generic keppra line. A critical feature of probe selection is the careful choice of probe sequence that is complementary to the sequence of the target of interest. Currently, nucleic acid probe hybrids are detected by incorporating pre-labeled probes in the methodology. Fluorescent-labeled probes offer the advantages of producing strong signals with less background, but have the disadvantages of poor ﬂuorescent signal stability and the purchase of a ﬂuorescence microscope with appropriate ﬁlters. Solid-phase hybridizations, such as line probe or dot blot assays, occur on a solid surface (nylon membranes) to which the nucleic acid probe is bound. In general, hybridizations assays on solid-support platforms are not as sensitive as those liquid-phase formats due to the lack of exposure to all target sequences. The controlled enzymatic digestion of cellular mem- branes and other proteins allow the probes to gain access to the target sequences. The labels for the nucleic acid probes, which can be biotin or digoxigenin, incorporate a signal compound, such as a colorimetric or a ﬂuorescent compound. Other formats have been designed to combine solution and solid- phased hybridization. In this model, the capture probes, which are coated on the metal beads, hybridize with the target nucleic acid in solution. A magnet is applied to the reaction tube, to separate the hybrids from the rest of the reaction and washing steps remove unbound probes and other unrelated molecules. In this solid support format, called “sandwich hybridization,” the signal probe will remain with the reac- tion only if the target is hybridized with both signal and capture probes. The sensitivity and speciﬁcity of probe hybridization formats are highly inﬂuenced by hybridization stringency conditions that occur during the reaction, such as the temperature settings, washing conditions, and formamide, pH, or salt buffer con- centrations. Assays with high stringency parameters are characterized by increased assay speciﬁcity and decreased sensitivity. Therefore high stringency parameters predict few hybrid mismatches and few false- positive results. In contrast, less stringent reactions increase the sensitivity of the assay at the risk of detecting unwanted, nonspeciﬁc results. In order to maximize assay performance characteristics, the stringency conditions for hybridization need to be optimized. Although most commercial hybridization assays are highly stan- dardized, a laboratory developed assay, may need to adjust hybridization conditions to achieve the level of stringency that ﬁts their needs. Clinical Application of Nonampli ﬁ ed Probe-Based Assays Probe-based assays have been developed to identify microbial nucleic acid targets from culture or directly from specimen. The accuracy, simplicity of use, and rapid turnaround time to results are advantages that have been applied to diagnostic test platforms, where they have been developed for the rapid identiﬁcation of a wide range of infectious agents, thus facilitating appropriate patient management and optimal treatment. The kits are nonisotopic, simple to use, and highly sensitive (92–100%) and speciﬁc (99–100%) (Table 12. The acridinium ester of unstable hybrids and unattached probes are degraded by alkaline hydrolysis thus preventing chemiluminescence. Assays for culture identiﬁcation of mycobacteria, include Mycobacterium tuberculosis complex, Mycobacterium kansasii , Mycobacterium gordonae, Mycobacterium avium complex, and speciﬁc probes to differentiate Mycobacterium intracellulare, from M. Probe assays are available for identiﬁcation of the following systemic dimorphic fungi: Blastomyces dermatitidis, Coccidioides immitis, 12 Nonampliﬁed Probe-Based Microbial Detection and Identiﬁcation 231 Table 12. In addition, tests for detection of the following bacteria directly from samples are available from Gen-Probe: Chlamydia trachomatis, N. The test is performed directly from a vaginal specimen without requiring nucleic acid ampliﬁcation. The technology includes distinct single-stranded nucleic acid capture probes and color development probes that are complementary to unique genetic sequences of each target organism. The capture probes are immobilized on a bead embedded in a Probe Analysis Card, along with separate beads for each target organ- ism. After hybridization and stringent wash steps, speciﬁc hybrids can be detected by colorimetric reactions. By visually comparing the hybridization pattern on the strip to a reference read-out template, the test result can easily be interpreted. Thus, the early identiﬁcation of these mutations may allow a timely adjustment of therapy to avoid hepatitis pro- gression [19 ]. Biotin is another popular label that can be detected with enzyme conjugates of avidin, streptavidin, or antibiotin antibodies. These enzymes convert soluble substrates into insoluble precipitates that appear as dark, localized cellular or subcellular stains. They hybridize to targets with improved high sensitivity and speciﬁcity mainly due to electrical neu- trality of its chemical structure. The rapid diagno- sis of bloodstream infections can signiﬁcantly improve patient care, management, and treatment by reducing turnaround time to 90 min, compared to 1–3 days by traditional culture-based methods. The assay was intended for detection from newly positive blood culture bottles that were smear-positive for yeast . Conventional culture-based identiﬁcation methods can lead to the initial administration of costly and inappro- priate antifungal therapy, whereas early species identiﬁcation enables optimal initial drug selection. An assay that identiﬁes staphylo- cocci from newly positive blood bottles with gram-positive cocci in clusters accurately differentiates S. Recently, 12 Nonampliﬁed Probe-Based Microbial Detection and Identiﬁcation 235 Table 12. Future Considerations Nonampliﬁed nucleic acid probes are successfully being used in clinical microbiol- ogy laboratories in many different formats. The technology will continue to expand due to their distinct advantages over culture-based tests, particularly in the detection and identiﬁcation of poorly growing pathogens and those with long generation times. The new probe-based assays under development will be designed to detect infec- tious agents directly from clinical specimens and ﬁxed tissue. Am J Pathol 141: 1247–1254 12 Nonampliﬁed Probe-Based Microbial Detection and Identiﬁcation 237 21. J Clin Microbiol 46:3470–3472 Chapter 13 Molecular Typing Techniques: State of the Art Richard V. Goering Introduction The treatment of infectious disease centers around the goals of both curing the patient and preventing or at least restricting the spread of disease. In a perfect world, health care professionals would know that these goals have been achieved when the patient’s health is restored and there are no new occurrences of infected patients. The individual patient may present with evidence of recurring or additional infection by a pathogen (e. Different members of a patient population may yield cultures of the same organism. In both instances, the question commonly asked is whether multiple isolates of a given pathogen represent the same strain. In the indi- vidual patient, this question commonly relates to issues of therapeutic efﬁcacy while in a patient population the concern is infection control. However, in both settings, the resolution of these questions is aided by speciﬁc epidemiological assessment. In the past, a variety of methods based on phenotypic characteristics have been used for this purpose including biotype, serotype, susceptibility to antimicrobial agents, or bacteriophages, etc. These included comparing protein molecular weight dis- tributions by polyacrylamide gel electrophoresis, relative mobility of speciﬁc enzymes by starch-gel electrophoresis (multi-locus enzyme electrophoresis), speciﬁc antibody reactions with immobilized cellular proteins (immunoblotting), and cellu- lar plasmid content (i. However, by the 1980s it was clear that comparisons at the genomic level would provide the most fundamen- tal measure of epidemiological relatedness. Goering While a wide range of etiological agents are of clinical concern, this review focuses on molecular approaches to the epidemiological analysis of bacterial pathogens. In any area of scientiﬁc investigation, state of the art methodology may be viewed from two different perspectives. There are cutting-edge techniques requiring spe- cialized equipment and expertise that perform remarkably well but are of limited availability to many investigators. Alternatively, there are functional state of the art approaches, meaning that one is using the best method available within the prevail- ing (ﬁnancial, geographic, technical expertise, etc. In this context, it is important to recognize that while one may not have access to the most recently published sophisticated methods, from an epidemiological standpoint, it is better to do something rather than nothing.
Provided there is not a tight fit between the bronchoscope and the airway buy keppra 250 mg low price, the risk of barotrauma is low keppra 500 mg cheap. The advantages of the Sanders system are that because continuous ventilation is possible (because the presence of an eyepiece is not necessary for ventilation of the lungs) buy keppra now, the duration of the bronchoscopy procedure is 2620 minimized order keppra 250mg free shipping, but the efficiency also permits extended bronchoscopy. Ventilation of the lungs may be achieved by connecting a mechanical ventilator to an anesthesia circuit that is connected to the bronchoscope side arm. One disadvantage of this ventilation technique is the presence of a leak of anesthesia gases, and consequentially, light anesthesia. Fiberoptic Bronchoscopy New generations of fiberscopes, with their improved optics and smaller diameters, have revolutionized bronchoscopy. Nasal fiberoptic bronchoscopy under topical anesthesia is well tolerated by most awake patients. The administration of an antisialagogue such as glycopyrrolate is useful in reducing secretions. Oral insertion is also possible in both awake and asleep patients and should be performed with a bite block in place to prevent damage to the bronchoscope. In all patients, insertion of the fiberoptic bronchoscope is associated with hypoxemia. The average decline in PaO2 is 20 mmHg and lasts for 1 to 4 hours after the procedure. This can be provided using mouth- held nasal prongs, a special face mask with a diaphragm through which the fiberscope can be passed, or an endotracheal tube with a T-piece diaphragm adapter. During and after fiberoptic bronchoscopy, patients experience increased 2621 airway obstruction. These changes are believed to be secondary to direct mechanical activation of irritative reflexes in the airway and, possibly, to mucosal edema. They may be avoided if atropine, either intramuscular or aerosolized into the airway, is administered before the procedure. If suction at 1 atm is applied to the fiberscope, air is removed at a rate of 14 L/min. The adult fiberscope can be passed through endotracheal tubes of 7 mm or greater internal diameter. Clearly, passage through an endotracheal tube decreases the cross-sectional area available for ventilating the patient, so if fibroscopy is planned, an endotracheal tube of the largest possible diameter should be used. A postendoscopy chest radiograph is advisable to exclude the presence of mediastinal emphysema or pneumothorax. In patients whose tracheas are intubated with endotracheal tubes of less than 7 mm internal diameter, use of pediatric fiberscopes, which have smaller diameters, would be more appropriate. The suction channel of the adult fiberoptic bronchoscope has been used to oxygenate and ventilate the lungs of patients. By attaching a jet ventilation system (similar to that used to drive the Sanders injector for rigid bronchoscopy) to the suction connection at the head of a fiberoptic bronchoscope, successful ventilation of the lungs of patients undergoing gynecologic procedures was achieved. This technique permitted adequate ventilation of patients with normally compliant lungs and chest walls. Ventilation of the lungs should be performed only with the tip of the instrument in the trachea because a more peripheral location may produce barotrauma. The lasers may be introduced into the bronchial tree through a fiberoptic bundle passed via the suction port of the fiberoptic bronchoscope. Complications of Bronchoscopy Complications of rigid bronchoscopy include mechanical trauma to the teeth, hemorrhage, bronchospasm, loss of a sponge, bronchial or tracheal perforation, subglottic edema, and barotrauma. Nevertheless, complications may arise owing to overdose with topical anesthetic, insertion trauma, local trauma, hemorrhage, upper airway obstruction related to passage of the instrument through an area of tracheal stenosis, hypoxemia, and bronchospasm. In most cases, it is best to intubate the trachea with an endotracheal tube after bronchoscopy under general anesthesia. This permits avoidance or treatment of some of these problems, particularly the increased airway irritability. Intubation also facilitates effective suctioning of the trachea and bronchi, and allows the patient to recover more gradually from general anesthesia. Diagnostic Procedures for Mediastinal Mass Patients with an anterior mediastinal mass may present a special problem for the anesthesiologist. Although such masses may cause obvious superior vena cava obstruction, they may also cause obstruction of major airways and cardiac compression, which are less obvious and may become apparent only on induction of anesthesia. Many cases of anesthetic-related airway compression from anterior mediastinal mass have been reported. In one case, total occlusion of the trachea starting 2 to 3 cm above the carina and extending to both main stem bronchi was observed, and a bronchoscope was passed through the obstruction. These findings suggested potential obstruction with onset of anesthesia; radiation therapy to the mediastinum was commenced, after which the flow–volume studies showed improved function. In a subsequent series of 105 patients with mediastinal masses, the incidence of intraoperative cardiorespiratory complications was 38%, and the incidence of 2623 postoperative respiratory complications was 11%. In another series of patients with mediastinal mass, four patients had abnormal spirometry but underwent general anesthesia without sequelae. However, a serious potential disadvantage of preoperative radiation therapy is that it may affect tissue histologic appearance, thereby preventing an accurate diagnosis. Furthermore, if the patient is a child, it may be difficult to obtain tissue samples under local anesthesia. No fatalities occurred in a series of 44 patients aged 18 years of age or younger with anterior mediastinal masses who underwent general anesthesia before radiation or chemotherapy. Preoperative evaluation of a patient with an anterior mediastinal mass to avoid life-threatening total airway obstruction is shown in Figure 38-21. If such obstruction occurs, it may be relieved by passage of a rigid bronchoscope or anode tube past the obstruction, by direct laryngoscopy,168 or by changing the position of the patient. Airway collapse and inability to ventilate has been reported in a previously asymptomatic patient with a mediastinal mass despite spontaneous ventilation with an inhaled anesthetic and an endotracheal tube. Positive-pressure ventilation was impossible, a rigid bronchoscopy was requested and the surgeons began to prepare femoral vessel access for emergent cardiopulmonary bypass. Fortunately, the airway patency was re-established when the patient’s spontaneous respiratory efforts improved as he awoke from general anesthesia. The authors emphasize the need for immediate availability of a rigid bronchoscope and that if a patient is at high risk, then serious 2624 consideration should be given to insertion of the femoral cannulas with cardiopulmonary bypass standing by before general anesthesia is induced. Cardiopulmonary bypass is not a suitable rescue modality unless the cannulae have been placed before induction because in the time required to achieve cannulation, severe neurologic damage is likely to occur. Thus during spontaneous inspiration, the normal transpulmonary pressure gradient distends the airways and helps maintain their patency, even in the presence of extrinsic compression. Mediastinoscopy Mediastinoscopy was introduced as a means of assessing spread of bronchial carcinoma. The lymphatics of the lung drain first to the subcarinal and paratracheal areas, and then to the sides of the trachea, the supraclavicular areas, and the thoracic duct. Examination of these nodes has provided a tissue diagnosis and greater selectivity of patients for thoracotomy. It is most useful in right lung tumors because left lung cancers tend to spread to subaortic nodes that are more accessible by an anterior mediastinoscopy in the second or third interspace (Chamberlain procedure). The anesthetic considerations for mediastinoscopy follow naturally from an understanding of the anatomy of this procedure and its potential complications. For cervical mediastinoscopy, the patient is placed in a reverse Trendelenburg (i. The instrument is advanced along the anterior aspect of the trachea and passes behind the innominate vessels and the aortic arch (Fig. The left recurrent laryngeal nerve is vulnerable as it loops around the aortic arch, and any of these structures may be traumatized. Because of scarring, previous mediastinoscopy may be considered a contraindication to a repeat examination. Relative contraindications include superior vena cava obstruction, tracheal deviation, and aneurysm of the thoracic aorta. Preoperative evaluation should include a search for airway obstruction or distortion. Evidence of impaired cerebral circulation, history of stroke, or signs of the Eaton–Lambert syndrome resulting from oat cell carcinoma should be sought. Blood must be available for the procedure because hemorrhage is a real risk and may be life- threatening. Most surgeons and anesthesiologists prefer general anesthesia using an endotracheal tube and continuous ventilation because this offers a more controlled situation and greater flexibility in terms of surgical manipulation. The anesthetic technique should include a muscle relaxant to prevent the patient from coughing because this may produce venous engorgement in the chest or trauma by the mediastinoscope to surrounding structures.
These patients often require radiologic procedures to determine the location and extent of the lesion prior to surgery purchase 250 mg keppra amex. These studies may then be used to program intraoperative localizing systems to help guide the surgical dissection order keppra 500mg with amex. Finally purchase 500 mg keppra, embolization of65 lesions may be performed a day or two prior to surgery to shrink the lesion and decrease blood loss during the operative procedure purchase keppra 500 mg amex. Prior surgical procedures may alter a previously easy airway, which subsequently may require advanced airway management techniques to intubate the trachea. Patients who have received cardiotoxic agents in which effects are dose dependent may require a cardiac evaluation including an echocardiogram. Decreased cardiac function may affect the type of anesthetic agents used and require invasive blood pressure monitoring with an arterial catheter. Prior treatment with neurotoxic agents may decrease the dose of muscle relaxants or cause their duration of action to be prolonged, requiring neuromuscular monitoring. Prior radiation therapy usually does not cause systemic problems unless the pituitary gland is damaged, which can give rise to the problems of panhypopituitarism leading to hypothyroidism, hypoadrenocorticism, and diabetes insipidus. Prior radiation therapy may lead to fibrosis and ankylosis in the temporomandibular joint, rendering direct laryngoscopy difficult. Previous radiation to the operative site may also increase blood loss and results in poor wound healing. Secondary radiation fibrosis may make the surgical dissection more difficult and time consuming. It may also necessitate the use of free or vascularized grafts to close the surgical site. In addition, the location of the donor site and potential anastomotic sites must be considered when positioning the patient. Close attention must be paid to the evaluation of the head and neck during the physical examination. Usually these patients do not have a difficult airway or require special techniques for intubation. For lesions requiring a midline surgical approach, oral intubation is the preferred route. Nasal endotracheal tubes may be secured by use of a heavy suture through the nasal septum and around the tube. Oral endotracheal tubes may be secured by wiring the endotracheal tube to the teeth, suturing it to the gingival periosteum, or using 3423 a circummandibular wire. There are two noteworthy points for anesthetic consideration: the use of muscle relaxation and blood conservation strategies. Similar to other procedures in which the facial nerve is at risk for injury or transection during dissection, it is necessary to avoid paralysis so that the facial nerve may be periodically stimulated to verify its integrity. Minimizing blood loss and creating a plan for replacement with blood products must be considered. If there is anticipation for large-volume blood loss, various approaches can be utilized to potentially reduce the need for transfusion. Acute normovolemic hemodilution can be used to minimize blood loss during the procedure. Recently, antithrombolytic therapy has been used with success in craniofacial procedures and may be of benefit in these cases. Blood salvage techniques, such as cell saver, are usually not appropriate given that most surgical sites are not reached through sterile approaches, and they would also be relatively contraindicated in surgeries involving resection of tumors. Upper Airway Infections Infectious processes of the upper airway can occur in the adult and present the same problems of airway compression, distortion, and compromise. Inflammation of the upper airway caused mainly by gram-negative bacteria may present with the same symptoms as epiglottitis in the pediatric age group. Although these patients present with fever, chills, drooling, and difficulty in speaking and swallowing, they do not usually appear with critical airways from swelling. These same symptoms may occur with Ludwig angina, which is a generalized cellulitis of the submandibular region. The infection69 is often the result of dental abscesses and extends into the submandibular, submental, and sublingual areas. Involvement of the sublingual spaces pushes the tongue upward and backward and can lead to asphyxiation due to obstruction of the airway. Should this occur, emergent surgical interventions may be required to drain the abscess and relieve the airway obstruction. Awake70 tracheostomy with local anesthesia has been considered the safest in these patients. If awake tracheostomy is performed, positive-pressure ventilation should be avoided until confirmation of proper tracheal tube placement, because insufflation into a false or blind passage can lead to significant patient morbidity. Alternative techniques of intubation include fiberoptic nasal intubation and direct laryngoscopy after inhalational 3424 anesthesia. The trachea should not be extubated until there is some resolution of the swelling. Maxillofacial Trauma Traumatic disruption of the bony, cartilaginous, and soft tissue components of the face and upper airway challenges the anesthesiologist to recognize the nature and extent of the injury and consequent anatomic alteration, create a plan for securing the airway safely, implement the plan without doing further damage, maintain the airway during the administration of an anesthetic, and determine when and how to extubate the patient’s trachea. Also necessary is the creation of a comfortable environment for both surgeon and anesthesiologist in a limited workspace. The lower third consists of the mandible, with its subdivisions of midline symphysis, body, angle, ramus, condyle, and coronoid process. The mandible has a unique, horseshoe shape that causes forces to gather at its points of vulnerability, often distant from the point of impact. Consequently, fractures of the mandible typically occur posteriorly where the cortex is also thinner—at the angle of the mandible, the ramus, and the condyle. Another common point of fracture is in the body of the mandible at the level of the first or second molar. Clinical experience indicates that this distribution occurs after high-velocity, high- impact trauma, such as occurs in an automobile accident. After trauma inflicted by a fist, a blunt weapon, or a fall, there is a greater tendency for a fracture of the symphysis, parasymphysis, and body to occur. The middle third contains the zygomatic arch of the temporal bone, blending into the zygomaticomaxillary complex, the maxillae, nasal bones, and orbits. Force from a blow to the midface, especially from in front and above, does not follow a normal vector of force dispersion and redistribution. Rather, it tends to create an abnormal shearing force, which may tear the facial skeleton from the cranial skeleton and extend the fracture into the base of the skull. Therefore, in any patient with severe midfacial trauma, a fracture of the base of the skull must be considered. The fracture segment may be displaced posteriorly or laterally or rotated about a vertical axis. The fracture crosses the medial wall of the orbit, including the lacrimal bone beneath the zygomaticomaxillary suture; crosses the lateral wall of the antrum; and passes posteriorly through the pterygoid plates. The line of fracture passes through the base of the nose and the ethmoid bone in its depth and through the orbital plates. The fracture line crosses the lesser wing of the sphenoid, then passes downward to the pterygomaxillary fissure and sphenopalatine fossa. From the base of the inferior orbital fissure, the fracture extends laterally and upward to the frontozygomatic suture and downward and backward to the root of the pterygoid plates. This apposition serves as a subtle clue to minimal posterior displacement of the midface. Foreign material from the nasopharynx may result in meningitis or, even more devastating, the endotracheal tube can enter the cranial cavity. Even positive- pressure bag and mask ventilation can force foreign material or air into the skull. Radiographic studies should be done prior to nasotracheal intubation74 whenever trauma to the skull base is suspected. One study revealed that in patients with maxillofacial injury due to low-velocity, low-impact blows, 4% had additional major life-threatening 3426 injuries and 10% had additional minor injuries. With high-velocity, high- impact accidents, 32% had major additional injuries and 31% had minor additional injuries. Multiple studies report cervical spine and significant head injury in patients with facial skeletal trauma, with incidence as high as 10.
Valve closure causes the second heart sound (S ); this event denotes2 end-systole order keppra online now. S is normally split because the pulmonic valve closes slightly2 after the aortic valve buy keppra without a prescription. During the first third of systolic ejection (the rapid ejection period) order 250mg keppra with visa, the curve of emptying is steep buy 250 mg keppra with amex. This final stage completes a single circuit (cardiac cycle) around the P–V diagram. The cardiac cycle proceeds in a time-dependent counterclockwise direction (arrows). Stroke volume and cardiac output are often substantially compromised as a result, leading to global tissue malperfusion. These three factors combine with the heart rate and rhythm to determine cardiac output. This phenomenon is known as the Bowditch, “staircase,” or “Treppe” effect, or the “force-frequency” relationship. Improved Ca2+ cycling efficiency and increased myofilament Ca2+ sensitivity are most likely responsible for the Bowditch effect. Maximal contractile force is generated at 150 to 180 stimulations/min in isolated cardiac muscle. These experimental data support the clinical observation of ideal matching of cardiac output to venous return at heart rates of approximately 175 beats/min during intense aerobic exercise in trained endurance athletes. Thus, tachyarrhythmias or rapid pacing may cause profound hemodynamic compromise, even in otherwise healthy individuals. Notably, the54 Bowditch effect is most likely of little consequence within a typical physiologic range of heart rate (e. This invasive technique is possible only in the cardiac catheterization laboratory or the operating room. This is not the case in many patients with significant pulmonary or cardiac disease. Afterload The additional load to which cardiac muscle is exposed after contraction has begun is termed “afterload. Power spectral or Fourier series analysis is used to calculate Zin(ω) from high-fidelity measurements of pressure and blood flow. Zin(ω) includes the frequency-dependent characteristics of the arterial vasculature, including its viscoelastic effects and wave reflection properties. This methodology is quite useful from a biomechanical engineering perspective, but has limited practical applicability. At end-systole, the64 forces driving further ejection and those resisting it are equal. Systemic vascular resistance primarily reflects the resistance of terminal arterioles, which is a major component of afterload. Wall thickening reduces (−) whereas chamber dilation increases (+) end-systolic wall stress as predicted by the Law of Laplace. Determining myocardial contractility is relatively easy in isolated cardiac muscle, but this measurement is remarkably difficult in the intact heart. Inotropic state and loading conditions are inextricably connected in the sarcomere and69 thus, it most likely not feasible to measure contractility as an independent variable. For example, dobutamine increases Ees, and the magnitude of this increase quantifies the positive inotropic effect of the drug. The slope of this “preload recruitable stroke work” relation has been shown to quantify changes in contractility in a relatively load-independent manner. However, this technique is rather time-consuming and is impractical when hemodynamics are unstable. All ejection phase indices of contractility are dependent on loading conditions and inotropic state. Ejection phase indices may also be inaccurate in the presence of mitral or aortic valve disease or a ventricular septal defect. In contrast, reductions in89 τ (indicative of more rapid relaxation) are observed during tachycardia, sympathetic nervous system activation, or administration of positive inotropic drugs. Development of heart failure causes characteristic changes in dV/dt morphology that are identical to the “delayed relaxation,” “pseudonormal,” and “restrictive” filling patterns measured using pulse wave Doppler (Fig. The relationship is also exponential such that σ = α(eβ − 1), where α is the coefficient of gain and β is the modulus of myocardial stiffness. A pulse95 wave Doppler echocardiography sample volume is placed between the tips of the mitral leaflets to obtain a high-resolution transmitral blood flow velocity envelope. As diastolic dysfunction worsens, a “pseudonormal” pattern replaces the “delayed relaxation” profile. Failure of a “restrictive” filling pattern to respond to administration of a vasodilator and revert to a “pseudonormal” or “delayed relaxation” pattern 785 carries a grim prognosis in patients with heart failure, unless a mechanical circulatory support device is implanted or cardiac transplantation is performed. This causes the second positive deflection (“D” [diastolic] wave) of the pulmonary venous blood flow velocity pattern. The presence of this blunted “S” wave allows the echocardiographer to distinguish between normal and “pseudonormal” transmitral blood flow velocity patterns because S/D < 1 in the latter condition. Profound blunting or reversal of the “S” wave under these circumstances indicates that moderate or severe mitral regurgitation is present, respectively. The reader is referred to Chapter 27 for a detailed discussion of the echocardiographic assessment of diastolic function. Pericardium The pericardium contains the heart, proximal great vessels, vena cavae, and pulmonary veins. The pericardium acts to separate the heart from other structures in the mediastinum and limits the heart’s movement through 787 its diaphragmatic and great vessel attachments. The fluid in the pericardium acts as a lubricant and consists of a combination of plasma ultrafiltrate, lymph, and myocardial interstitial fluid (total volume of 15 to 35 mL). The pericardium is much less compliant than myocardium, and has very limited volume reserve (Fig. While the pericardium is acutely noncompliant, a slow increase in pericardial pressure (e. This compensatory response increases the pericardium’s compliance and reduces its restraining forces, thereby allowing the heart to continue functioning without imminent hemodynamic collapse. Note that large increases in pericardial volume occur after reserve volume is exceeded. The hemodynamic consequences of ventricular interdependence form the basis of respiratory cycle-induced pulse pressure and stroke volume variation, which have been shown to the useful dynamic indices of preload in conscious and anesthetized patients. It is especially important to appreciate that spontaneous ventilation is crucial in these conditions because negative intrathoracic pressure assists venous return, whereas cardiovascular collapse may occur with the initiation of positive pressure ventilation because venous return becomes profoundly limited. Effect of heart failure on the mechanism of exercise-induced augmentation of mitral valve flow. Intraoperative transesophageal echocardiography for surgical repair of mitral regurgitation. Effects of pacing-induced and balloon coronary occlusion ischemia on left atrial function in patients with coronary artery disease. The effects of right ventricular apical pacing on ventricular function and dyssynchrony implications for therapy. Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms. Molecular diversity of myofibrillar proteins: gene regulation and molecular significance. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of β-agonist stimulation. Structure of the actin-myosin complex and its implications for muscle contraction. Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the prepower stroke state. Determinants of left ventricular filling and of the diastolic pressure-volume relation.