Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics . These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified . In this study , we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen . After 8 days , rings compacted to < 1 % of original volume and cell number increased 2.4 fold . We initiated continuous cyclic stretching of the rings after 2 , 4 , or 8 days of incubation , while monitoring the tissue forces . Peak tissue force during each cycle decreased rapidly after initiating stretch , followed by further slow decline . We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix . Cell force was estimated by subtracting matrix force from tissue force . After 12 h , matrix force-strain curves were highly nonlinear . Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior . Cell stiffness increased with stretching frequency from 0.001 -- 0.25 Hz . Cell stiffness decreased with stretch amplitude -LRB- 5 -- 25 % -RRB- at 0.1 Hz . The trends in cell stiffness do not fit simple viscoelastic models previously proposed , and suggest possible strain-amplitude related changes during cyclic stretch . INTRODUCTION Mechanical stimuli are important for the health and maintenance of many biological functions .14 This is especially true in the cardiovascular system where the tissues and organs are undergoing constant cyclic loading . It is the cellular response to these mechanical forces that can either maintain healthy tissue or lead to a pathologic state .1 Mechanical stimuli have been used to create more functionally viable engineered tissues .2 -- 5,9,15 -- 17 It has been shown that constant amplitude cyclic stretch -LRB- a form of mechanical conditioning -RRB- of engineered tissue constructs can cause cells to reorient , increase cell proliferation , and produce more extracellular matrix -LRB- ECM -RRB- .3 However , these studies generally have looked at how the tissue responds to mechanical conditioning over extended time .9,15 In addition , the studies generally quantify the mechanics using only the ultimate strength and modulus4 ,5,16 along with quantification of such biological factors as cell number , gene expression , and ECM components .2,3,17 Since it is the cells that actively respond to mechanical loads , it is important to understand the effects of mechanical conditioning on the individual components of the tissue -LRB- cell and matrix -RRB- and how the mechanical properties change from the time that the conditioning is initiated . Our earlier work has shown that tissue forces drop with subsequent stretches during repeated cyclic stretching .19 However , whether this drop occurs in the cells and/or matrix components is not known . Furthermore , it is not known how these drops are affected by different amplitudes and frequencies over many hours of continuous cyclic stretch . The objective of this study is to measure the short-term -LRB- < 24 h -RRB- mechanical response of collagen gels populated with cardiac fibroblasts to cyclic stretch at different amplitudes and frequencies . Although engineered tissue constructs may be composed of several cell and matrix components , we chose to use a simple system composed of a single cell type and a single defined ECM component to study the mechanics of tissue components during cyclic stretch . This system provides a 3D environment for the cells while allowing the components to be easily modified and quantified . The benefits of using collagen , the main structural component in cardiovascular tissue , are that its initial concentration can be easily modified and it is more stable than other ECM components such as fibrin .4,5,10 By using a single cell type we can avoid complicating effects due to mixed cell populations and clearly understand that cell type 's contribution to the tissue mechanics . Cardiac fibroblasts are one of the two main cell types in cardiac tissue and are responsible for ECM maintenance and remodeling .7 These cells , along with the cardiac myocytes , undergo continuous cyclic stretch with each heartbeat . Thus , it is particularly useful to understand their cellular mechanics during cyclic stretch . By measuring the cell forces under various cyclic stretch amplitudes and frequencies , we can better understand the relationship between the applied strains and the cells ' mechanical response . Then we can model this behavior and be able to predict cellular responses to dynamic physiologic and pathologic loading conditions . METHODS Tissue Ring Fabrication Chicken embryonic cardiac fibroblasts -LRB- CECF -RRB- were isolated from 10 day old chicken embryos -LRB- Charles River Laboratories , Wilmington , MA -RRB- . Cells were maintained in Dulbecco 's modified Eagle 's medium -LRB- DMEM -RRB- supplemented with 3 % fetal bovine serum -LRB- FBS -RRB- , 50 units/ml penicillin and 50 mg/ml streptomycin -LRB- P/S -RRB- . Tissues were fabricated as described previously .20 Briefly , CECF from passage 2 -- 8 were added to a collagen solution containing monomeric rat tail tendon collagen -LRB- Upstate Biotechnology Inc. , Lake Placid , NY -RRB- neutralized with 0.1 N sodium hydroxide -LRB- NaOH -RRB- and 2 × concentrated DMEM . The final solution contained 1 mg/ml collagen and one million cells/ml . Then 1 ml of this solution was poured into cylindrical molds containing an inner mandrel -LRB- diameter = 9.55 mm -RRB- . After one hour of incubation , the solution gelled and the molds were filled with incubation medium containing DMEM supplemented with 10 % FBS and P/S . After five additional hours , the gels were removed from the molds and transferred to spacers -LRB- Fig. 1 -RRB- placed in incubation medium . The spacers consisted of two fixed rods -LRB- 2.07 mm diameter -RRB- separated by a distance to maintain the internal circumference of the rings at 30 mm . Medium was changed every other day . FIGURE 1 . Cardiac fibroblast populated rings on spacers during static incubation -LRB- A -RRB- Day 0 -LSB- 6 h -RSB- -LRB- B -RRB- Day 2 -LRB- C -RRB- Day 4 -LRB- D -RRB- Day 8 . Mean ring width was 0.64 mm after 8 days incubation -LRB- see Fig. 4 -RRB- . FIGURE 2 . Illustration of peak force versus time . The gray curve is sample data obtained for 10 stretch cycles . The black line plots the peak force for each cycle vs. time . This peak force line is used for many of the following figures . Mechanical Tests After 2 , 4 , or 8 days of static incubation -LRB- Fig. 1 -RRB- the width of the rings was measured and then the rings were removed from the spacers . The rings were then mounted on a mechanical tester described previously20 that allows independent programmable stretching of four rings while monitoring applied force . The test medium contained DMEM HEPES modification supplemented with 10 % FBS and P/S . All rings were placed on the tester at a baseline length that maintained the inner circumference of 30 mm . This was defined as 0 % strain . In early tests , stretching began immediately after mounting the rings . This led to increases in peak force after initiation of stretch for low amplitudes at early incubation times . Upon further study , it was determined this was a result of the fresh media and FBS used in the test baths and not a result of a mechanical stretch induced response . In all further studies , the rings were mounted on the tester and held at baseline length for 3 h to allow them to reach equilibrium before testing began . Effect of Cyclic Stretch Amplitude Four rings were tested at each of the three days with each ring stretched either 5 % , 10 % , 20 % , or 25 % at 0.1 Hz using a triangular displacement waveform -LRB- constant strain rate during loading -RRB- . The force was continuously monitored at 10 Hz over 14 h. For many of the figures , the peak force , defined as the maximum force during each cycle , is plotted vs. time -LRB- Fig. 2 -RRB- . We use the term stretch to refer to the amplitude of cyclic stretching , while we use the term strain to refer to the amount of deformation at a particular point in the loading curve . To separate out the individual tissue component forces -LRB- cell and matrix -RRB- , 2 μM Cytochalasin D -LRB- CytoD -RRB- was added either 3 h before or 12 h after initiation of stretch . CytoD disrupts actin filaments inside the cell and thus eliminates the majority of the cell-contributed force .21 The remaining force measured 120 min after addition of CytoD is termed the matrix force . Then the estimated cell force is calculated by taking the matrix force and subtracting that from the total tissue force without CytoD . FIGURE 3 . Cell force calculation . Matrix force was measured by adding 2μM CytoD to rings prior to initiation of cyclic stretch . Cell force is calculated by taking the normalized matrix force -LRB- see Eq . 1 -RRB- and subtracting it from the tissue force . To estimate the cell forces during the initial 12 h of cyclic stretch , it is necessary to use two separate rings subject to identical stretching protocols . This is accomplished by subtracting the forces measured in a ring with CytoD added prior to cyclic stretching from the forces measured in a ring where CytoD was added after 12 h of cyclic stretch . We denote these forces FC0 -LRB- t -RRB- and FC12 -LRB- t -RRB- respectively and similarly , we denote the peak forces PC0 -LRB- t -RRB- and PC12 -LRB- t -RRB- . However , the peak forces after 14 h of cyclic stretch , when both rings have been treated with CytoD , can still differ between rings subject to identical cyclic stretch protocols due to variations between rings . To account for this variation , we scale the forces in the ring to which CytoD was added before initiating stretch using the following formula : We found that the shape of the loading/unloading curve was similar whether CytoD was added before or after initiation of cyclic stretch . We then subtracted this scaled , or normalized , force curve from the force curve for the ring with CytoD added after 12 h to obtain an estimated cell force -LRB- Fig. 3 -RRB- : Single Step Stretch and Hold Effect To compare the effect of a single stretch to cyclic stretching , a step stretch -LRB- stretch to a given stretch magnitude in less than 0.1 s -RRB- of 5 % , 10 % , or 20 % was applied and the rings were held at this stretched length for 8 h while the force was monitored . The tissue was then returned to its baseline length and the force was monitored for an additional 6 h . In another set of experiments , CytoD was added 3 h before or 12 h after either a 10 % or 20 % step stretch . Effect of Cyclic Stretch Frequency A similar procedure was followed to test frequency effects . Four rings were tested at each of the three days with each ring stretched at either 0.25 Hz , 0.1 Hz , 0.01 Hz , or 0.001 Hz at 10 % stretch amplitude . The current tester 's upper frequency limit is 0.25 Hz and thus unable to stretch at higher frequencies that are closer to physiologic values . CytoD was added either 3 h before or 12 h after initiation of stretch to separate out the cell and matrix components as a result of different frequencies of cyclic stretch . Cell Number After the completion of testing , some rings were washed in phosphate buffered saline -LRB- PBS -RRB- and then placed in 1 ml lysis buffer -LRB- 0.1 % sodium dodecyl sulfate -LRB- SDS -RRB- in PBS -RRB- . The samples were sonicated until the rings completely disintegrated to release the DNA from the cells . Finally , 30 μl of sample was placed in 3 ml of Hoechst working solution -LRB- 30 nM Hoechst 33258 dye -LRB- Sigma , St. Louis , MO -RRB- in PBS -RRB- . The fluorescence was then read by a spectrophotometer and cell number determined from a standard curve obtained from samples with known numbers of cells . Immunohistochemistry Rings were removed from spacers after 2 , 4 , or 8 days static incubation and immediately placed in a solution of 4 % paraformaldehyde for 30 min , washed with PBS and then permeabilized for 45 min in 0.2 % Triton X-100 in PBS . This was followed by 1 h incubation in a blocking solution -LRB- 2 % normal goat serum and 0.02 % sodium azide in PBS -RRB- . After blocking , the rings were incubated overnight at 4 °C in blocking solution containing TRITC phalloidin . Finally , the rings were washed again in PBS and mounted for viewing on a confocal microscope -LRB- Zeiss Confocor -RRB- . RESULTS Ring Width Ring width was used as a metric for monitoring the extent of compaction during incubation . The initial width of the rings was approximately 10 mm . This width decreased until day 8 -LRB- Fig. 4 -LRB- b -RRB- -RRB- when the average width was reduced to 0.64 mm . At this time the cross-sectional area of the ring appeared circular and the volume could be approximately calculated using a circular cross-sectional area and the circumference of the ring . A ring diameter of 0.64 mm corresponds to a 99 % reduction in the initial tissue volume . FIGURE 4 . Cell number and ring width during static incubation . -LRB- A -RRB- Cell number increases from the initial 1 million cells to 2.4 million cells after 8 days of static incubation . -LRB- B -RRB- Ring width decreased from an initial 10 mm to 0.64 mm after 8 days of static incubation , with a large amount of compaction occurring prior to day 2 . Cell Number Rings started with a cell density of one million cells/ml -LRB- one million cells per ring -RRB- . This number increased steadily for 4 days to 2.3 million cells then increased only slightly to day 8 with a total cell number of 2.4 million -LRB- Fig. 4 -LRB- a -RRB- -RRB- . When combined with the 99 % reduction in tissue volume , this translates into a final cell density of 234 million cells/ml or a 234 fold increase in cell density . Amplitude Effect The effect of increasing stretch amplitude on the peak tissue and matrix forces is shown in Fig. 5 . At all amplitudes , the peak tissue forces immediately after initiating stretch -LRB- Fig. 5 -LRB- a -RRB- -RRB- increased with static incubation time while the peak matrix forces were similar -LRB- Fig. 5 -LRB- c -RRB- -RRB- . After 12 h of cyclic stretch , both tissue -LRB- Fig. 5 -LRB- b -RRB- -RRB- and matrix forces had decreased substantially , with a larger percentage decline in the matrix force -LRB- Fig. 5 -LRB- d -RRB- -RRB- . To examine the relationships between tissue , matrix , and cell forces more closely , figures in the remainder of this paper show data from rings statically incubated for 8 days prior to cyclic stretch unless otherwise noted . Similarities or differences to days 2 and 4 are discussed in the text . In addition , all curves are averages across repeated -LRB- n = 2 -- 7 -RRB- experiments . Due to the large number of different conditions tested and the relatively low number of samples -LRB- exact numbers shown in Fig. 5 -LRB- e -RRB- , 10 -LRB- e -RRB- -RRB- rigorous statistical analysis was not possible . Tissue Force Higher stretch amplitudes resulted in higher initial peak forces -LRB- Figs. 5 -LRB- a -RRB- , 6 -LRB- a -RRB- , 6 -LRB- b -RRB- -RRB- . With each subsequent stretch , the peak force decreased with large drops during the first several stretches and sustained decreases during the first two hours , but only small further decreases in peak force over the next 10 h -LRB- Fig. 6 -LRB- a -RRB- -RRB- . In addition , this decrease in peak force was greater at higher stretch amplitudes both in terms of absolute force drop and percentage drop from first stretch . At days 2 and 4 , after 12 h of stretch , higher amplitudes still have higher peak forces but the differences were smaller between the amplitudes than the first stretch differences -LRB- Fig. 5 -LRB- a , b -RRB- -RRB- . FIGURE 5 . Peak tissue and matrix forces for different cyclic stretch amplitudes at 0.1 Hz . -LRB- A -RRB- Peak tissue force of the first cyclic stretch after 2 , 4 , or 8 days static incubation . Peak tissue force increases with cyclic stretch amplitude and incubation time . -LRB- B -RRB- Peak tissue force after 12 h of cyclic stretch after 2 , 4 , or 8 days static incubation . At day 8 the peak tissue force is similar regardless of cyclic stretch amplitude . -LRB- C -RRB- Peak matrix force of the first stretch after 2 , 4 , or 8 days static incubation . Peak matrix force increases with cyclic stretch amplitude . -LRB- D -RRB- Peak matrix force after 12 h of cyclic stretch after 2 , 4 , or 8 days static incubation . Higher amplitudes result in higher peak forces . -LRB- E -RRB- Number of rings averaged for data shown in Figures 5-8 at each amplitude and static incubation day . -LRB- F -RRB- Individual measurements used in panel B -LSB- Peak tissue force after 12 h , 8 days of incubation -RSB- . This shows that the trend within an experiment , i.e. rings fabricated on a single day , is consistent but the experiment to experiment variation in forces is large . Y-axis scales are different in each panel to emphasize the trends with amplitude and incubation time.FIGURE 6 . Tissue force versus cyclic stretch amplitude after 8 days of static incubation . -LRB- A -RRB- Peak tissue force over 12 h of cyclic stretch . Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline . -LRB- B -RRB- First 5 tissue stretches after initiation of cyclic stretch . A large decline in tissue force and hysteresis occurred between the first and second stretch . -LRB- C -RRB- Last 5 tissue stretches after 12 h of cyclic stretch . The peak tissue force is similar at the four amplitudes . By day 8 , similar peak tissue forces were seen after 12 h of cyclic stretch regardless of stretch amplitude between 5 % and 25 % -LRB- Figs. 5 -LRB- b -RRB- , 6 -LRB- a -RRB- , 6 -LRB- c -RRB- -RRB- . The rings subject to lower stretch amplitudes -LRB- 5 % or 10 % -RRB- were able maintain a positive baseline force -LRB- i.e. force at the baseline , or zero strain , length -RRB- . At the highest stretch amplitude the rings had periods of zero force during both the loading and unloading of the ring , meaning that at low strains the rings were slack -LRB- Fig. 6 -LRB- c -RRB- -RRB- . After the first stretch -LRB- Fig. 6 -LRB- b -RRB- -RRB- , the loading curve remained nearly linear at 5 % stretch amplitude , but became increasingly nonlinear at higher stretch amplitudes -LRB- Fig. 6 -LRB- c -RRB- -RRB- . FIGURE 7 . Matrix force versus cyclic stretch amplitudes after 8 days of static incubation . -LRB- A -RRB- Peak matrix force over 12 h of cyclic stretch . Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12 h duration . -LRB- B -RRB- First 5 matrix stretches after initiation of cyclic stretch . A large decline in matrix force and hysteresis occurred between the first and second stretch . -LRB- C -RRB- Last 5 matrix stretches after 12 h of cyclic stretch . Matrix loading curve is highly nonlinear and peak force increased slightly with higher cyclic stretch amplitude.FIGURE 8 . Cell force versus cyclic stretch amplitudes after 8 days of static incubation . -LRB- A -RRB- Peak cell force over 12 h of cyclic stretch . Peak cell force increased for 5 % and 10 % amplitudes while it decreased for 20 % and 25 % amplitudes . -LRB- B -RRB- First 5 cell stretches after initiation of cyclic stretch . -LRB- C -RRB- Last 5 cell stretches after 12 h of cyclic stretch . The peak cell force and cell stiffness decreased with increased amplitude . The loading curves are nearly linear for all amplitudes.FIGURE 9 . Response to step stretch and hold after 8 days of static incubation . The tissue , matrix , and cell forces are shown after a step stretch of either 10 % or 20 % . The tissue is held at the stretched length for 12 h . The tissue and matrix force for a 20 % step and hold is higher than that for a 10 % step and hold . The cell force is identical for 10 % and 20 % . Matrix Force Except for the first stretch , the matrix loading curves were nonlinear for all stretch amplitudes tested . Similar to tissue force , higher stretch amplitudes led to higher initial peak forces -LRB- Figs. 5 -LRB- c -RRB- , 7 -LRB- b -RRB- -RRB- . For all amplitudes , the peak force decreased with repeated cyclic stretching and this drop in peak force was larger at higher stretch amplitudes -LRB- Fig. 7 -LRB- a -RRB- -RRB- but higher stretch amplitudes still maintained higher peak forces -LRB- Fig. 7 -LRB- c -RRB- -RRB- . For the first stretch , the ratio of peak forces at day 8 for 25 % to 5 % was 5:1 , but this ratio decreased to 2:1 after 12 h of stretching , due to the peak matrix force dropping by a greater percentage at the higher amplitudes over time . The other distinct feature was the shape of the loading/unloading curves . After several hours of stretching the shapes of the loading/unloading curves were very similar for all stretch amplitudes , but the strain value at which the matrix force began increasing from zero during each loading cycle was shifted toward larger strain values . The amount of this shift was greater for higher stretch amplitudes -LRB- Fig. 7 -LRB- c -RRB- -RRB- . This meant that the matrix contributed to the tissue force during the loading only when the strain had reached a value from 3 to 7 % less than its maximum value . This shape and shift is evident at days 2 , 4 , and 8 . For all stretch amplitudes , the matrix is slack at low strains . Cell Force Cell force increased with increased incubation time . The increase in cell force after 12 h of stretch from 2 to 4 days of incubation was due to an increase in cell number -LRB- per cell force remained the same -RRB- . However , the increase in cell force from days 4 to 8 could not be explained by the increase in cell number alone , indicating an increase in per cell force . The shape of the first force-strain curve for the cell differed from all subsequent curves . The initial baseline force was followed by a rise and then a leveling off with further increases in force seen only at the higher amplitudes -LRB- Fig. 8 -LRB- b -RRB- -RRB- . During the second stretch , the cell force loading curve was nearly linear . Unlike the tissue and matrix , the cell force loading curve retained a nearly linear shape throughout the 12 h of cyclic stretch even at 25 % amplitude -LRB- Fig. 8 -LRB- b , c -RRB- -RRB- . At day 2 , the peak cell force initially drops for several cycles , but then gradually increases to the end of the test . The size of the initial force drop is dependent on the stretch amplitude . The trend is similar at day 4 , except the cell force at 25 % stretch remains fairly steady instead of increasing . At day 8 , the peak cell force , after the initial drop , increases only for the 5 % and 10 % stretch , while it decreases for the 20 % and 25 % stretch -LRB- Fig. 8 -LRB- a -RRB- -RRB- . Single Step Stretch & Hold Effect Similar to cyclic stretching at different amplitudes , the larger magnitude step stretches led to higher initial forces -LRB- Fig. 9 -RRB- . The tissue force was higher than the matrix force immediately after the step at the same magnitude . However , both tissue and matrix force dropped quickly and to a greater extent for higher magnitudes . The matrix force was lower than the tissue force during the entire 12 h hold period at the same magnitude indicating that the cells contributed to the measured tissue force throughout the hold period -LRB- Fig. 9 -RRB- . The rate of decrease during the 12 h hold period is similar for both the tissue and matrix . This is evident in the fact that very quickly after the step stretch the cell force approaches a constant for the 12 h -LRB- Fig. 9 -RRB- . The cell force is approximately the same at both 10 % and 20 % step stretches during the 12 h held at the stretched length . For both step stretch magnitudes , the cell force at the end of the 12 h hold period is similar to the cell force at the baseline length -LRB- data not shown -RRB- immediately before the step stretch is applied . Rings that did not have CytoD added before or during the step stretch were returned to their baseline lengths after being held at the stretched length for 8 h . The force dropped to zero but then began to recover with lower magnitude step stretches increasing their forces faster than higher magnitude step stretches . The recovered forces approached their baseline forces prior to stretch . After 6 h of monitoring force at baseline , rings stretched to 5 % magnitude and held recovered more of their baseline force -LRB- 84 % -RRB- than rings stretched to 20 % magnitude -LRB- 42 % -RRB- . The force decreases more rapidly in the step stretch and hold than the peak force drops in cyclic stretching at the same amplitude . Frequency Effect Figure 10 illustrates the trends in the peak tissue -LRB- Fig. 10 -LRB- a , b -RRB- -RRB- and matrix force -LRB- Fig. 10 -LRB- c , d -RRB- -RRB- either at the first stretch or after 12 h of cyclic stretch . It also compares the peak force trends over stretch frequencies and static incubation time . At all frequencies the peak tissue and matrix force increased with static incubation -LRB- Fig. 10 -RRB- . FIGURE 10 . Peak tissue and matrix forces for different cyclic stretch frequencies at 10 % amplitude . -LRB- A -RRB- Peak tissue force of the first cyclic stretch after 2 , 4 , or 8 days static incubation . Peak tissue force increased with cyclic stretch frequency and incubation time . -LRB- B -RRB- Peak tissue force after 12 h of cyclic stretch after 2 , 4 , or 8 days static incubation . At day 8 the peak tissue force increased slightly with increased frequency . -LRB- C -RRB- Peak matrix force of the first stretch after 2 , 4 , or 8 days static incubation . Peak matrix force increased slightly with increased cyclic stretch frequency . -LRB- D -RRB- Peak matrix force after 12 h of cyclic stretch after 2 , 4 , or 8 days static incubation . Peak matrix force similar regardless of cyclic stretch frequency . -LRB- E -RRB- Number of rings averaged for data shown in Figs. 10 -- 13 at each frequency and static incubation day . -LRB- F -RRB- Individual measurements used in panel B -LSB- Peak tissue force after 12 h and 8 days of static incubation -RSB- . This shows that the trend within an experiment -LRB- E # -RRB- is consistent but the experiment to experiment variation is large . Y-axis scales are different in each panel to emphasize the trends with frequency and incubation time . Tissue Force Initially , higher frequencies led to higher peak tissue forces -LRB- Figs. 10 -LRB- a -RRB- , 11 -LRB- a -RRB- , 11 -LRB- b -RRB- -RRB- . After an initial drop , the peak force remained steady or increased at days 2 and 4 . However , at day 8 after the initial drop the peak force continues to decline over the rest of the experiment -LRB- Fig. 11 -LRB- a -RRB- -RRB- . After 12 h , higher frequencies still resulted in higher peak forces -LRB- Figs. 10 -LRB- b -RRB- , 11 -LRB- c -RRB- -RRB- . Another difference is in the baseline forces , where lower frequencies had higher baseline forces than high frequencies . Thus , rings cyclically stretched at lower frequencies have lower measured tissue stiffness -LRB- slope of the loading curve -RRB- . FIGURE 11 . Tissue force versus cyclic stretch frequency at day 8 . -LRB- A -RRB- Peak tissue force over 12 h of cyclic stretch . Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline . -LRB- B -RRB- First 5 tissue stretches after initiation of cyclic stretch . A large decline in tissue force occurred between the first and second stretch . -LRB- C -RRB- Last 5 tissue stretches after 12 h of cyclic stretch . The tissue stiffness increased with increasing cyclic frequency . Matrix Force Initially , higher frequencies led to higher peak matrix forces -LRB- Figs. 10 -LRB- c -RRB- , 12 -LRB- a -RRB- , 12 -LRB- b -RRB- -RRB- . These matrix forces quickly dropped over the first few hours and then continued a steady decline for the remainder of the cyclic stretch -LRB- Fig. 12 -LRB- a -RRB- -RRB- . At day 8 , all the peak matrix forces quickly approached the same value about 30 min after the start of stretch -LRB- Fig. 12 -LRB- a -RRB- -RRB- . In addition , unlike the tissue and cell curves , there is little difference between the matrix curves at the different frequencies after 12 h except that lower frequencies contribute over a slightly larger range of strains but the peak force is still the same at all frequencies tested -LRB- Fig. 12 -LRB- c -RRB- -RRB- . As with the matrix force at different amplitudes , the loading curve was nonlinear and the matrix contributed only to the tissue force when the strain was within 4 -- 6 % of its maximum value . FIGURE 12 . Matrix force versus cyclic stretch frequency at day 8 . -LRB- A -RRB- Peak matrix force over 12 h of cyclic stretch . Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12 h duration . -LRB- B -RRB- First 5 matrix stretches after initiation of cyclic stretch . A large decline in matrix force occurred between the first and second stretch . -LRB- C -RRB- Last 5 matrix stretches after 12 h of cyclic stretch . Matrix loading curves were highly nonlinear and the peak forces was similar at all frequencies . Cell Force Similar to the first force-strain curves at different amplitudes , the cell force initially rose , then leveled off or even decreased at the highest frequency -LRB- Fig. 13 -LRB- b -RRB- -RRB- . At lower frequencies the slope of the loading curve was lower than that for higher frequencies -LRB- Fig. 13 -LRB- c -RRB- -RRB- . This is clear at day 8 when , for low frequency , the baseline cell force was higher and the peak cell force was lower than that of high frequencies -LRB- Fig. 13 -LRB- c -RRB- -RRB- . At the two lowest frequencies the cells were able to maintain a baseline force while at the two highest frequencies the baseline cell force was near zero . This leads to a cell stiffness that was higher at higher cyclic stretch frequencies . This was similar to the trend seen with the tissue stiffness . FIGURE 13 . Cell force versus cyclic stretch frequencies at day 8 . -LRB- A -RRB- Peak cell force over 12 h of cyclic stretch . -LRB- B -RRB- First 5 cell stretches after initiation of cyclic stretch . -LRB- C -RRB- Last 5 cell stretches after 12 h of cyclic stretch . Cell stiffness increased with increasing cyclic stretch frequency . Immunohistochemistry Rings stained with a dye for actin cytoskeleton showed a distinct morphology that developed during the 8 days of incubation . Initially the cells were sparse with few cells on the outer layer of the rings -LRB- Fig. 14 -LRB- a -RRB- -RRB- . The inner core of the rings initially showed a higher cell concentration that had some alignment along the circumferential direction -LRB- Fig. 14 -LRB- b -RRB- -RRB- . As the rings incubated , this outer layer that was approximately 25 μm thick increased in density but showed little preferential alignment -LRB- Fig. 14 -LRB- c , e -RRB- -RRB- . The inner core of the rings also had some increase in cell density but was noticeably less dense than the outer layer -LRB- Fig. 14 -LRB- d , f -RRB- -RRB- . However , these cells were highly aligned in the circumferential direction -LRB- Fig. 14 -LRB- d , f -RRB- -RRB- . FIGURE 14 . Confocal images at 2 , 4 , and 8 days of static incubation . F-actin fibers visualized using a TRITC phalloidin stain . -LRB- A -RRB- Outer surface -LRB- projection from 4 -- 14 μm depth -RRB- after 2 days static incubation . -LRB- B -RRB- Inner core -LRB- projection from 150 -- 160 μm depth -RRB- after 2 days static incubation . -LRB- C -RRB- Outer surface -LRB- projection from 4 -- 14 μm depth -RRB- after 4 days static incubation . -LRB- D -RRB- Inner core -LRB- projection from 61 -- 71 μm depth -RRB- after 4 days static incubation . -LRB- E -RRB- Outer surface -LRB- projection from 10 -- 15 μm depth -RRB- after 8 days static incubation . -LRB- F -RRB- Inner core -LRB- projection from 60 -- 65 μm depth -RRB- after 8 days static incubation . Field of View : 230 μm × 230 μm . Circumferential direction is in the vertical direction . DISCUSSION We have shown that a few hours after the initiation of stretch , higher cyclic stretch amplitude does not lead to higher cell or tissue forces . Thus , for the majority of the cyclic stretch duration , the peak stress experienced by the cells is independent of the stretch amplitude applied , at least in the range of 5 % -- 25 % . This is accomplished by the cells becoming less stiff at higher stretch amplitudes . The exact mechanism for this change in cell stiffness is unknown . We have found that the magnitude of the drop in tissue force after the addition of 2 μm CytoD is similar to the force drop after adding detergent -LRB- e.g. deoxycholate -RRB- . Since the detergent treatment actually removes the cells ,18 it is likely the actin cytoskeleton is primarily responsible for the change in cell stiffness . Whether this is a result of cytoskeletal remodeling and/or reduction in active contraction/relaxation of the myosin is not known . While the initial extracellular matrix consists entirely of collagen it is reasonable to assume that over 8 days of incubation time that additional cell-contributed matrix may be produced . We have incubated rings for 20 days prior to cyclic stretching and found that the matrix force-strain curves are similar to those after 8 days of incubation . Peak forces increase only a small amount at 5 % or 10 % stretch amplitude after 14 h of cyclic stretch at 0.1 Hz . These results suggest that the matrix mechanics do not change substantially even in the presence of cells that can produce additional matrix . In addition , we treated some rings with 30 mM ribose after 10 days of incubation , and incubated them for an additional 10 days to non-enzymatically crosslink the collagen .9 This treatment increased the peak matrix force during cyclic stretch with a 3 to 4 fold increase after 14 h compared to the untreated rings incubated for 20 days . The shapes of the matrix force-strain curves changed somewhat , with a shorter period of slack during each force-strain curve . The increased peak force with glycation was similar to Isenberg and Tranquillo9 but comparing the shapes of the force-strain curves is difficult due to differences in experimental methods . We also found that increased matrix stiffness due to crosslinking had little effect on cell force for 5 % or 10 % cyclic stretch amplitude at 0.1 Hz . Cell force-strain curves were almost identical for treated and untreated rings after 12 h of cyclic stretch . In 2-D cultures , cell traction depends upon the stiffness of the substrate .6 The cellular mechanism for this response is not clear , but Discher and co-workers suggest that both `` outside-in '' and `` inside-outside-in '' pathways may be involved . Whether 3-D culture conditions or cyclic stretching reduce the influence of matrix stiffness on cellular mechanics remains to be investigated . Since it is the cells that are responding to the mechanical conditioning it is important to realize that while the cells are undergoing a higher strain they are not undergoing a higher stress . So if the cells respond to stress , higher stretch amplitudes will not increase this response . Since both the cells and the tissues at different stretch amplitudes maintain similar stresses , it is reasonable to assume the cells are responding to maintain a certain range of mechanical stresses . Some limitations to estimating the cell-contributed force should be noted . Unlike the tissue and matrix force we are not able to measure the cell force directly but instead have to estimate it based on the direct measurement of the tissue and matrix . In addition , to estimate the cell force at more than a single time point , we must use the forces from different rings . Due to the ring to ring variations , it is necessary to normalize one ring to another to get quantitative values for the cell force . However , because this normalization occurs at 12 h , it is unknown how well this single point normalization works in predicting the cell force 12 h earlier during the initial stretches . Furthermore , the assumption that the cell force is simply the subtraction of the matrix force from the tissue force is only valid after a critical cell density is reached .11 At days 2 and 4 it is unlikely that this critical cell density is reached but it is surpassed based on cell density by day 8 . Unlike the cells that respond to increased stretch amplitude by decreasing their stiffness , the matrix force-strain curves shift so that the matrix contributes only when the strain is within 3 % -- 7 % of the maximum value with the matrix contributing over a slightly larger range of strains at higher stretch amplitudes . This response of the matrix does not appear to depend upon the cells having an actin cytoskeleton , since we found a similar response when cytoD is added either before stretch is initiated or after 12 h of stretch . After 12 h of cyclic stretch , the peak matrix force is only slightly higher with increased stretch amplitude . With different frequencies of cyclic stretch , the peak matrix force is similar but the matrix contributes over a larger range of strains at lower frequencies . For a 10 % stretch , the matrix contributes to the last 6 % of the stretch at 0.001 Hz while contributing only to the last 4 % at 0.25 Hz . The higher baseline tissue and cell forces seen at lower frequencies -LRB- 0.001 Hz and 0.01 Hz -RRB- are consistent with a simple viscoelastic model because the tissues and cells have more time to recover before being stretched again . The same is seen in the lower amplitudes at 0.1 Hz -LRB- 5 % and 10 % -RRB- which are able to maintain a baseline force . These correspond to the lowest strain rates and may allow for recovery . For a linear viscoelastic material undergoing continuous cyclic stretch , both higher stretch amplitudes and higher strain rates will lead to higher peak forces . For cyclic stretch at constant frequency , the highest stretch amplitude also corresponds to the highest strain rate . However , we observed that the peak cell force decreased as the amplitude -LRB- and the strain rate -RRB- increased . We also note that the decrease in peak cell force is a function of both stretch amplitude and strain rate because similar strain rates -LRB- 5 % / s -RRB- at different amplitudes -LRB- e.g. 10 % @ 0.25 Hz and 25 % @ 0.1 Hz -RRB- show different tissue , matrix and cell responses In the case of the matrix at 25 % @ 0.1 Hz , there is no positive matrix force until the strain increases beyond 10 % ; whereas there is matrix force below 10 % strain during cyclic stretch at 10 % @ 0.25 Hz . To explain this complex behavior a model of cellular mechanics that includes strain-dependent and strain rate-dependent damage may be required . It is reasonable to assume large and/or quick deformations may result in disruption of the actin cytoskeleton8 leading to damage , and thus a reduction in the cell force and stiffness . However , any damage model will be complicated by viscoelastic effects . One possible mechanism for a strain dependent change in stiffness is an alteration in the amount of cell activation through a stretch-activated pathway . Two of the pathways that exist to activate myosin-light chain kinase and thus force generation in fibroblasts are a calcium dependent pathway and a Rho dependent pathway .13 To test the possible involvement of a calcium dependent pathway , we tested some rings in the presence of 50 μM or 500 μM of gadolinium chloride -LRB- GdCl3 -RRB- to block stretch activated calcium channels .12 In the presence of 50 μM of GdCl3 , day 2 rings showed a decrease in peak tissue force at both 5 % and 10 % cyclic stretch compared to untreated controls . This peak force decrease continued for the first 5 -- 6 h of cyclic stretch then began to increase for the remainder of the 12 h of cyclic stretching but still did not return to the peak tissue force of untreated controls . However , for day 8 rings neither 50 nor 500 μM GdCl3 had an effect on the tissue force compared to untreated controls . Since GdCl3 reduces the tissue and cell force during the first several hours of cyclic stretch at day 2 , it suggests that the calcium dependent pathway plays a role in maintenance of cell force at least during early tissue development . It is unclear what causes the cell force to increase after the initial decrease but it could be due to an upregulation of calcium independent pathways or incomplete inhibition of new or existing stretch activated channels . The lack of an effect of GdCl3 at day 8 suggests a reduced role of the calcium dependent pathway . In addition , these results have important implications for the tissue engineering field , especially for those tissues that undergo continuous deformations such as in the cardiovascular system . By isolating the cell contribution to tissue force , we are able to provide a better understanding of the mechanical environment of the cell and aid in designing mechanical conditioning protocols that improve tissue properties . For example , the peak cell force is not constant during cyclic stretch and thus cell stimulation varies with time . Furthermore , if cell response is dependent on cell stress or force , increasing the amplitude of conditioning will not increase and may decrease the applied cell force . Due to the large changes in mechanical behavior after initiation of cyclic stretch , it is important to characterize engineered tissues ' response to these loading conditions over longer time periods . Current mechanical characterization relies mostly on a few cycles of preconditioning followed by a test to failure .4,5,9,16 While failure tests are necessary , the response of the tissue and cells under physiologic loading is also vital . We have shown that mechanical properties may change dramatically over hours after initiation of stretch . These changes have implications on tissue function in vivo after implantation and these changes can not be captured in simple failure tests . CONCLUSIONS We have shown that bioartificial tissue constructs consisting of a single cell type and matrix component are a useful model system in which to explore tissue , matrix , and cell mechanics . The cell response to cyclic stretch is complex and depends on both the amplitude and frequency of cyclic stretch . The change in cell stiffness with amplitude behavior does not fit current cellular mechanics models . Future investigations may explore the microstructural basis for this behavior .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tissue engineering
cell mechanics
cardiac fibroblasts
collagen


Hippocampal

sclerosis

dementia

differs

from

hippocampal

sclerosis

in

frontal

lobe

degeneration

Hippocampal

sclerosis

-LRB-

HS

-RRB-

is

characterized

by

selective

neuronal

loss

and

gliosis

in

and

the

subiculum

and

has

been

associated

with

several

disorders

including

Alzheimer

's

disease

frontotemporal

lobar

degeneration

with

ubiquitin

immunoreactive

inclusions

-LRB-

FTLD-U

-RRB-

vascular

dementia

and

some

tauopathies

In

some

cases

HS

is

not

associated

with

other

degenerative

pathologies

Such

cases

are

sometimes

referred

to

as

HS

dementia

-LRB-

HSD

-RRB-

Differences

between

HSD

and

HS

in

the

setting

of

FTLD-U

have

not

been

systematically

investigated

To

this

end

eight

cases

of

HSD

and

ten

cases

of

HS

associated

with

FTLD-U

were

studied

with

Nissl

and

periodic

acid-Schiff

stains

to

assess

neuronal

loss

and

corpora

amylacea

respectively

Sections

were

immunostained

with

antibodies

to

glial

fibrillary

acidic

protein

HLA-DR

and

synaptophysin

and

immunoreactivity

was

measured

with

image

analysis

in

and

the

subiculum

of

each

case

Additionally

sections

were

immunostained

with

antibodies

to

tau

to

determine

the

presence

of

argyrophilic

grains

HSD

was

different

from

HS

associated

with

FTLD-U

Specifically

it

was

more

common

in

the

elderly

and

it

was

associated

with

more

marked

neuronal

and

synaptic

loss

and

with

greater

reactive

gliosis

Corpora

amylacea

tended

to

be

more

frequent

in

HSD

than

in

FTLD-U

but

there

was

no

difference

in

frequency

of

argyrophilic

grains

Introduction

Hippocampal

sclerosis

-LRB-

HS

-RRB-

is

defined

as

selective

neuronal

loss

and

gliosis

of

and

the

subiculum

of

the

hippocampus

Its

prevalence

in

demented

patients

ranges

from

to

-LSB-

-RSB-

It

is

associated

with

several

other

disorders

including

Alzheimer

's

disease

-LRB-

AD

-RRB-

frontotemporal

lobar

degeneration

with

ubiquitin

immunoreactive

inclusions

-LRB-

FTLD-U

-RRB-

vascular

dementia

dementia

with

Lewy

bodies

and

some

tauopathies

Occasionally

HS

has

been

reported

as

an

independent

pathologic

explanation

for

dementia

the

so-called

HS

dementia

-LRB-

HSD

-RRB-

The

reported

prevalence

of

HSD

ranges

from

to

-LSB-

-RSB-

The

etiology

of

HS

is

uncertain

in

most

cases

but

it

is

hypothesized

to

be

due

to

either

hypoxic-ischemic

injury

or

to

neurodegeneration

of

selectively

vulnerable

neuronal

populations

in

and

the

subiculum

-LSB-

-RSB-

An

hypoxic-ischemic

origin

is

supported

by

the

known

deficient

hippocampal

vascular

supply

-LSB-

-RSB-

and

the

fact

that

hippocampal

neurons

have

a

higher

susceptibility

to

hypoxia

due

to

their

concentration

of

glutamate

receptors

which

may

be

involved

in

excitotoxic

cell

death

-LSB-

-RSB-

Further

arguments

in

favor

of

this

theory

are

the

association

of

hypoxic-ischemic

episodes

in

patients

with

HS

-LSB-

-RSB-

and

the

increased

prevalence

of

cardiac

-LSB-

-RSB-

and

cerebrovascular

disease

-LSB-

-RSB-

in

cases

of

HS

of

presumed

hypoxic-ischemic

origin

Since

many

cases

of

HS

do

not

have

histories

of

hypoxia

or

ischemia

-LSB-

-RSB-

cardiac

disease

-LSB-

-RSB-

or

cerebrovascular

accidents

-LSB-

-RSB-

a

degenerative

cause

is

also

proposed

This

hypothesis

is

supported

by

the

frequent

association

of

HS

with

degenerative

dementias

like

AD

-LSB-

-RSB-

and

FTLD-U

-LSB-

-RSB-

and

the

less

frequently

with

tauopathies

mainly

argyrophilic

grain

disease

-LRB-

AGD

-RRB-

and

-LSB-

-RSB-

The

aim

of

this

study

was

to

compare

the

pathological

characteristics

of

HSD

versus

HS

in

the

setting

of

FTD-U

Materials

and

methods

The

Mayo

Clinic

Jacksonville

brain

bank

database

between

and

of

individuals

with

dementia

or

degenerative

neuropathology

-LRB-

N

-RRB-

was

screened

for

specimens

with

HS

that

had

complete

neuropathologic

evaluations

This

evaluation

included

description

of

gross

and

microscopic

findings

as

well

as

quantitative

information

about

Alzheimer

type

pathology

Specifically

the

reports

include

counts

of

senile

plaques

and

neurofibrillary

tangles

in

six

cortical

sections

four

sectors

of

the

hippocampus

two

regions

of

the

amygdala

as

well

as

subcortical

regions

with

thioflavin-S

fluorescent

microscopy

It

also

involves

immunohistochemistry

for

tau

α-synuclein

and

ubiquitin

as

appropriate

as

previously

described

-LSB-

-RSB-

A

Braak

neurofibrillary

tangle

stage

was

assigned

to

all

cases

based

upon

the

distribution

of

neurofibrillary

tangles

with

thioflavin-S

fluorescent

microscopy

as

previously

described

-LSB-

-RSB-

HS

was

detected

in

cases

in

cases

-LRB-

-RRB-

it

was

associated

with

a

primary

degenerative

disease

process

mostly

AD

-LRB-

men

and

women

years

of

age

-RRB-

and

FTLD-U

-LRB-

men

and

women

years

of

age

P

compared

to

AD

-RRB-

HS

was

detected

in

about

of

AD

cases

-LRB-

of

-RRB-

and

of

FTLD-U

cases

-LRB-

of

-RRB-

The

remainder

of

cases

included

HS

in

the

setting

of

a

wide

range

of

pathologies

including

Lewy

body

disease

progressive

supranuclear

palsy

corticobasal

degeneration

and

multiple

system

atrophy

In

eight

cases

HS

was

not

associated

with

degenerative

pathology

For

this

study

cases

were

selected

for

further

study

and

placed

in

one

of

two

groups

The

first

group

of

eight

cases

was

operationally

termed

HSD

most

HSD

cases

-LRB-

-RRB-

were

associated

with

evidence

of

cerebrovascular

disease

When

compared

to

a

control

group

of

cases

from

the

same

brain

bank

matched

for

age

sex

and

Braak

stage

cerebrovascular

disease

was

detected

in

of

the

controls

a

frequency

similar

to

that

in

HSD

Therefore

we

could

not

necessarily

assume

that

the

HS

was

due

to

hypoxic-ischemic

causes

In

all

HSD

cases

FTLD-U

was

ruled

out

with

ubiquitin

immunohistochemistry

The

second

group

-LRB-

FTLD-U

HS

-RRB-

included

cases

of

HS

associated

to

FTLD-U

of

the

FTLD-U

HS

cases

had

pathologic

evidence

of

cerebrovascular

disease

or

other

pathologic

processes

and

HS

was

assumed

to

be

related

to

FTLD

In

all

cases

other

pathologic

processes

such

as

progressive

supranuclear

palsy

corticobasal

degeneration

or

Lewy

body

disease

were

absent

Alzheimer

type

pathology

was

minimal

of

the

cases

in

either

the

HSD

or

FTLD-U

HS

group

had

a

Braak

neurofibrillary

tangle

stage

greater

than

IV

In

of

the

cases

could

neuronal

loss

in

the

hippocampus

be

attributed

to

neurofibrillary

tangles

since

had

many

extracellular

-LRB-

ghost

-RRB-

neurofibrillary

tangles

Medial

temporal

tauopathy

due

to

AGD

was

not

a

basis

for

exclusion

and

all

cases

were

screened

for

this

age-related

pathology

with

immunohistochemistry

using

isotype-specific

tau

antibodies

as

previously

reported

-LSB-

-RSB-

Coronal

sections

of

the

posterior

hippocampus

at

the

level

of

the

lateral

geniculate

nucleus

were

cut

at

a

thickness

of

μm

Sections

were

stained

with

Nissl

and

periodic

acid-Schiff

-LRB-

PAS

-RRB-

stains

Sections

were

immunostained

on

a

DAKO

Autostainer

with

antibodies

to

HLA-DR

glial

fibrillary

acidic

protein

-LRB-

GFAP

-RRB-

tau

and

synaptophysin

using

procedures

similar

to

those

reported

previously

-LSB-

-RSB-

The

following

primary

antibodies

were

used

anti-GFAP

-LRB-

monoclonal

Biogenex

San

Ramon

CA

-RRB-

anti-HLA-DR

-LRB-

monoclonal

eBioscience

San

Diego

CA

-RRB-

tau

-LRB-

monoclonal

Dr.

Peter

Davies

Albert

Einstein

College

of

Medicine

Bronx

NY

-RRB-

and

anti-synaptophysin

-LRB-

monoclonal

Dr.

Peter

Davies

Albert

Einstein

College

of

Medicine

Bronx

NY

-RRB-

For

antigen

retrieval

slides

were

steamed

in

distilled

water

for

min

For

immunostaining

which

has

been

shown

to

be

a

sensitive

and

specific

method

for

detecting

AGD

-LSB-

-RSB-

slides

were

also

pretreated

in

formic

acid

for

min

The

degree

of

neuronal

loss

in

the

subiculum

-LRB-

end

plate

-RRB-

and

dentate

gyrus

was

evaluated

on

Nissl

stained

sections

using

a

three-point

rating

scale

absent

mild-to-moderate

and

marked

PAS

staining

was

used

to

assess

the

density

of

corpora

amylacea

Corpora

amylacea

were

defined

as

round

PAS-positive

structures

in

the

neuropil

perivascular

subpial

and

subependymal

areas

-LSB-

-RSB-

Presence

of

corpora

amylacea

throughout

the

hippocampus

was

graded

on

a

four-point

scale

absent

sparse

moderate

and

many

To

assess

astrocytic

microglial

and

synaptic

immunoreactivity

sections

immunostained

for

GFAP

HLA-DR

and

synaptophysin

were

used

Standardized

non-overlapping

digital

images

of

the

pyramidal

layer

of

and

subiculum

were

taken

under

a

objective

on

a

light

microscope

Immunoreactivity

was

quantified

as

a

percentage

area

using

MetaMorph

software

version

-LRB-

Molecular

Devices

Sunnyvale

CA

-RRB-

Four-R

-LRB-

-RRB-

tau

immunostained

slides

were

used

to

determine

the

presence

of

AGD

Cases

were

considered

to

have

AGD

if

they

had

small

dot

or

comma-like

argyrophilic

lesions

in

neuronal

processes

of

the

pyramidal

layer

and

the

entorhinal

cortex

as

well

as

coiled

bodies

in

temporal

white

matter

which

are

the

same

criteria

used

in

other

studies

-LSB-

-RSB-

Clinical

and

neuropathologic

records

were

reviewed

to

collect

data

on

age

sex

Braak

neurofibrillary

tangle

stage

brain

weight

and

macroscopic

and

microscopic

neuropathology

findings

at

autopsy

The

recorded

gross

findings

at

autopsy

were

lobar

hippocampal

and

mammillary

body

atrophy

The

remarkable

microscopic

findings

were

vascular

pathology

ischemic

brain

injury

and

white

matter

disease

Differences

between

the

two

groups

in

age

Braak

stage

brain

weight

corpora

amylacea

neuronal

loss

and

glial

and

synaptic

immunoreactivity

pathology

were

analyzed

with

unpaired

t

test

and

Mann

Whitney

U

tests

depending

upon

the

variable

Fisher

's

exact

test

was

used

to

compare

sex

presence

of

AGD

and

remarkable

pathologic

findings

The

statistical

analyses

were

performed

using

Sigma

Stat

for

Windows

version

-LRB-

Systat

Software

Richmond

CA

-RRB-

and

the

significance

levels

were

set

at

P

Results

A

summary

of

the

demographic

and

neuropathologic

findings

of

the

cases

included

in

this

study

is

presented

in

Table

A

comparison

of

the

two

groups

is

presented

in

Table

The

cases

with

HSD

had

an

older

age

of

death

than

those

with

FTLD-U

HS

-LRB-

vs.

P

-RRB-

In

the

HSD

group

six

of

eight

cases

were

men

while

four

of

six

cases

in

the

FTLD-U

HS

group

were

men

but

this

difference

was

not

significant

Table

and

neuropathologic

features

of

HS

casesAgeSexNFT

stageNeuronal

lossVascular

pathologyCerebrovascular

angiopathyLacunar

infarctsMicroscopic

infarctsIschemic

-LRB-

PCA

-RRB-

PV

WM

BG

Th

F

M

BG

ThO

PV

-LRB-

ICA

MCA

-RRB-

WM

BGF

P

OBG

pons

WM

BG

ThPV

F

O

-LRB-

MCA

PCA

ACA

-RRB-

BG

CBO

Cortical

WSACortical

WSA

O

WM

BG

PV

WM

BG

amygdala

ThPV

F

T

F

P

F

T

P

F

T

F

F

T

F

T

subcortical

and

F

T

subcortical

and

F

P

F

T

P

subcorticalSub

subiculum

CA

cornu

ammonis

EP

endplate

DG

dentate

gyrus

F

frontal

T

temporal

P

parietal

O

occipital

M

motor

ICA

internal

carotid

artery

ACA

anterior

cerebral

artery

MCA

middle

cerebral

artery

PCA

posterior

cerebral

artery

VA

vertebral

artery

BA

basilar

artery

PV

periventricular

DC

deep

cerebral

WM

white

matter

BG

basal

ganglia

Th

thalamus

CB

cerebellum

WSA

watershed

area

not

available

P

represent

HSD

cases

F#

represent

FTLD-U

HS

casesTable

of

HSD

and

FTLD-U

HSHSD

-LRB-

n

-RRB-

FTLD-U

HS

-LRB-

n

-RRB-

Age

at

death

-LRB-

years

-RRB-

mean

Std

Dev

Sex

-LRB-

of

cases

-RRB-

NFT

stage

median

-LRB-

tile

tile

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Brain

weight

-LRB-

g

-RRB-

mean

Std

Dev

Gross

atrophy

-LRB-

of

cases

-RRB-

Frontal

Medial

temporal

Lateral

temporal

loss

-LRB-

score

-RRB-

median

-LRB-

tile

tile

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Synaptophysin

-LRB-

area

-RRB-

mean

SEMSubiculum

GFAP

-LRB-

area

-RRB-

mean

SEMSubiculum

-LRB-

area

-RRB-

mean

Corpora

amylacea

-LRB-

score

-RRB-

median

-LRB-

tile

tile

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Argyrophilic

grain

disease

-LRB-

of

cases

-RRB-

exact

test

was

used

for

categorical

variables

t

test

for

continuous

variables

and

Mann

Whitney

Rank

Sum

test

for

discontinuous

variables

P

P

Neuropathologic

findings

The

Braak

neurofibrillary

tangle

stage

was

significantly

greater

in

HSD

than

in

FTLD-U

HS

The

brain

weight

was

also

greater

in

HSD

than

in

FTLD-U

HS

Cortical

atrophy

was

more

frequent

in

FTLD-U

HS

than

HSD

in

frontal

-LRB-

vs.

-RRB-

medial

temporal

-LRB-

vs.

-RRB-

lateral

temporal

-LRB-

vs.

-RRB-

and

parietal

lobes

-LRB-

vs.

-RRB-

The

difference

reached

statistical

significance

in

frontal

and

medial

temporal

regions

Both

groups

showed

marked

gross

atrophy

of

the

hippocampal

formation

and

mammillary

body

In

this

comparative

study

cerebrovascular

pathology

was

virtually

limited

to

the

HSD

group

Atherosclerosis

of

large

vessels

at

the

base

of

the

brain

was

present

in

seven

of

eight

HSD

cases

Three

had

minimal

had

mild

-LRB-

occluding

less

than

of

the

lumen

-RRB-

and

had

moderate

atherosclerosis

-LRB-

occluding

between

and

of

the

lumen

-RRB-

Arteriosclerosis

defined

by

presence

of

hyalinosis

of

the

media

and

adventitial

fibrosis

of

small

-LRB-

μm

diameter

-RRB-

vessels

was

present

in

-LRB-

-RRB-

cases

and

cerebral

amyloid

angiopathy

-LRB-

CAA

-RRB-

was

present

in

-LRB-

-RRB-

of

HSD

Three

-LRB-

-RRB-

cases

of

FTLD-U

HS

had

minimal

atherosclerosis

of

the

cases

of

this

group

had

significant

arteriolosclerosis

or

evidence

of

CAA

Ischemic

brain

injury

was

present

in

six

of

eight

HSD

cases

but

in

of

the

FTLD-U

HS

cases

The

eight

HSD

cases

included

-LRB-

-RRB-

with

lacunar

infarcts

-LRB-

-RRB-

with

microscopic

infarcts

and

-LRB-

-RRB-

with

microscopic

foci

of

ischemic

gliosis

Most

cases

independent

of

their

group

showed

white

matter

pathology

defined

by

rarefaction

and

attenuation

of

white

matter

associated

with

dilated

perivascular

spaces

loss

of

myelinated

fibers

and

gliosis

These

findings

were

present

in

the

periventricular

white

matter

in

seven

of

eight

HSD

cases

In

three

of

these

cases

more

diffuse

white

matter

pathology

was

also

present

involving

the

centrum

semiovale

All

of

the

cases

with

FTLD-U

HS

showed

white

matter

pathology

that

followed

the

distribution

of

the

cortical

atrophy

Two

of

the

HSD

cases

-LRB-

and

-RRB-

had

only

minimal

atherosclerosis

and

CAA

which

was

probably

insufficient

to

cause

hippocampal

hypoperfusion

but

both

cases

had

a

history

of

coronary

artery

disease

and

congestive

heart

failure

which

might

account

for

hypoxic-ischemic

injury

to

the

hippocampus

although

this

remains

purely

speculative

since

these

risk

factors

are

common

in

individuals

of

this

age

without

HS

-LSB-

-RSB-

Another

case

of

HSD

-LRB-

-RRB-

with

minimal

atherosclerosis

arteriosclerosis

and

CAA

had

a

history

of

epilepsy

as

well

as

laminar

ischemic

gliosis

in

the

watershed

cortices

and

cortical

cystic

infarcts

and

thalamic

ischemic

gliosis

Epilepsy

in

this

patient

was

probably

secondary

to

a

porencephalic

cyst

that

was

present

in

the

temporal

pole

Given

the

absence

of

degenerative

pathology

this

case

was

included

in

the

HSD

group

but

presence

of

extensive

cerebrovascular

disease

raises

the

possibility

that

HS

in

this

case

was

related

to

hypoxic-ischemic

injury

Quantitative

and

semi-quantitative

analyses

Both

HSD

and

FTLD-U

HS

had

severe

neuronal

loss

in

the

pyramidal

layer

of

and

the

subiculum

No

significant

differences

were

found

in

the

severity

of

neuronal

loss

between

the

two

regions

nevertheless

differences

in

the

distribution

of

neuronal

loss

were

noted

Most

cases

had

neuronal

loss

in

both

and

the

subiculum

except

for

two

cases

of

HSD

that

had

isolated

involvement

and

two

cases

of

FTLD-U

HS

that

had

isolated

subicular

involvement

The

region

and

the

endplate

were

not

affected

in

most

cases

Only

cases

of

HSD

occasionally

had

neuronal

loss

in

these

regions

with

involvement

of

in

and

involvement

of

and

endplate

in

The

dentate

was

involved

in

half

of

the

cases

of

both

groups

Synaptic

loss

assessed

by

image

analysis

of

density

of

synaptophysin

immunoreactivity

was

significantly

less

in

region

in

HSD

compared

to

FTLD-U

HS

-LRB-

Fig.

-RRB-

A

similar

trend

was

noted

in

the

subiculum

Gliosis

as

assessed

by

image

analysis

with

GFAP

and

HLA-DR

immunostains

tended

to

be

greater

in

HSD

compared

with

FTLD-U

HS

-LRB-

Fig.

-RRB-

although

it

did

not

reach

statistical

significance

probably

because

of

small

sample

size

Both

GFAP

and

HLA-DR

immunoreactivity

were

greater

in

HSD

than

in

FTLD-U

HS

in

both

and

the

subiculum

Fig.

of

region

of

hippocampus

in

HS

associated

with

FTLD-U

-LRB-

a

c

e

-RRB-

and

HSD

-LRB-

b

d

f

-RRB-

immunostained

for

synaptophysin

-LRB-

a

b

-RRB-

GFAP

-LRB-

c

d

-RRB-

and

HLA-DR

-LRB-

e

f

-RRB-

Note

better

preservation

of

neuropil

and

less

gliosis

in

FTLD-U

HS

compared

to

HSD

-LRB-

All

figures

are

-RRB-

There

tended

to

be

more

corpora

amylacea

in

HSD

than

in

FTLD-U

HS

but

this

did

not

reach

statistical

significance

A

concomitant

diagnosis

of

AGD

was

made

in

two

cases

of

FTLD

HS

and

in

one

case

of

pure

HS

-LRB-

-RRB-

Discussion

The

present

study

confirmed

the

characteristic

findings

of

neuronal

loss

and

gliosis

in

and

the

subiculum

in

HS

regardless

of

etiology

however

there

were

some

significant

differences

between

HSD

and

HS

that

occurs

in

FTLD-U

In

addition

there

were

subjective

differences

that

did

not

meet

statistical

significance

probably

related

to

the

small

sample

size

in

this

study

The

results

support

the

hypothesis

that

there

are

differences

in

underlying

disease

mechanisms

in

HSD

compared

to

HS

in

FTLD-U

From

a

demographic

perspective

demented

patients

with

HS

have

been

shown

to

be

older

than

demented

patients

without

HS

-LSB-

-RSB-

although

there

are

exceptions

-LSB-

-RSB-

Data

concerning

the

age

distribution

among

patients

with

HS

has

to

our

knowledge

not

been

reported

In

the

present

study

we

show

HSD

to

mainly

affect

the

elderly

-LRB-

years

of

age

-RRB-

as

opposed

to

HS

in

FTLD-U

The

younger

age

of

the

FTLD-U

may

account

for

the

lower

frequency

of

concurrent

cerebrovascular

disease

since

an

age

sex

and

Braak

neurofibrillary

tangle

stage-matched

cohort

had

comparable

frequency

of

cerebrovascular

disease

Microscopically

there

were

three

important

differences

in

the

pathology

in

HSD

compared

with

HS

in

FTLD-U

First

HSD

had

lower

synaptophysin

immunoreactivity

suggesting

a

more

destructive

process

resulting

in

more

severe

synaptic

loss

while

in

FTLD-U

there

is

selective

neuronal

loss

but

relative

preservation

of

the

neuropil

presumably

from

preserved

afferent

and

efferent

projections

to

sectors

that

show

neuronal

loss

Second

the

difference

in

the

distribution

of

neuronal

loss

suggested

subicular

involvement

to

be

characteristic

of

HS

in

FTLD-U

Even

though

the

differences

in

distribution

of

neuronal

loss

did

not

reach

statistical

significance

other

observations

support

this

idea

-LSB-

-RSB-

Third

HSD

was

associated

with

greater

astrocytic

and

microglial

reaction

compared

to

HS

in

FTLD-U

Glial

activation

has

been

proposed

to

be

caused

by

neuronal

damage

which

is

present

in

both

ischemic

and

degenerative

pathology

but

may

be

exacerbated

in

HSD

due

to

more

extensive

tissue

damage

affecting

not

only

neurons

but

also

the

neuropil

Macroscopically

FTLD-U

HS

had

more

severe

cortical

atrophy

a

not

surprising

finding

since

it

correlates

with

FTLD

-LSB-

-RSB-

HSD

on

the

other

hand

showed

a

pattern

of

atrophy

often

localized

to

the

hippocampal

formation

arguing

against

this

pathology

being

associated

with

more

widespread

cortical

degeneration

This

fits

with

results

from

previous

studies

of

HSD

that

showed

that

loss

of

synaptophysin

immunoreactivity

in

the

hippocampus

was

not

accompanied

with

similar

loss

in

the

cortex

or

basal

ganglia

-LSB-

-RSB-

Corpora

amylacea

have

been

proposed

as

a

possible

marker

for

hypoxic-ischemic

HS

since

they

may

be

numerous

in

brains

of

patients

exposed

to

repetitive

hypoxic

episodes

-LSB-

-RSB-

They

have

also

been

proposed

as

a

useful

marker

for

evaluating

HS

in

surgical

specimens

removed

for

treatment

of

temporal

lobe

epilepsy

-LSB-

-RSB-

Although

there

was

a

trend

for

more

corpora

amylacea

in

HSD

than

in

FTLD-U

HS

it

did

not

reach

statistical

significance

which

may

be

further

evidence

against

the

argument

that

HSD

is

due

to

hypoxic-ischemic

injury

Even

though

we

did

not

find

a

difference

in

the

frequency

of

AGD

in

HSD

and

HS

associated

with

FTLD-U

the

presence

of

AGD

in

of

the

cases

may

suggest

an

association

of

HS

with

AGD

as

reported

in

previous

studies

-LSB-

-RSB-

This

study

included

two

cases

of

HSD

-LRB-

and

-RRB-

in

which

there

was

not

only

no

evidence

of

degenerative

pathology

but

also

no

evidence

of

cerebrovascular

pathology

Cases

in

which

HS

is

the

only

pathologic

explanation

for

dementia

are

rare

Hattanpa

found

some

cases

of

HS

to

be

false

negative

cases

for

FTLD-U

after

further

studying

them

with

ubiquitin

immunohistochemistry

-LSB-

-RSB-

Since

we

analyzed

our

cases

with

ubiquitin

false

negatives

were

excluded

giving

a

probable

explanation

for

the

lower

prevalence

compared

to

the

and

previously

reported

by

others

-LSB-

-RSB-

The

two

cases

of

HSD

with

neither

degenerative

nor

cerebrovascular

pathology

had

cardiovascular

risk

factors

that

could

link

HS

to

an

ischemic

condition

of

systemic

origin

They

are

similar

to

the

ones

that

have

been

reported

by

others

-LSB-

-RSB-

On

the

other

hand

in

a

systematic

survey

of

cardiovascular

risk

factors

including

electrocardiograph

abnormalities

congestive

heart

failure

and

cardiomegaly

by

imaging

and

autopsy

studies

there

was

no

increased

frequency

of

these

findings

in

HSD

compared

to

age-matched

controls

without

HS

-LSB-

-RSB-

The

present

study

confirms

the

finding

that

HS

in

the

setting

of

dementia

in

most

cases

should

be

regarded

as

a

feature

linked

to

a

concurrent

degenerative

disease

process

particularly

FTLD-U

-LSB-

-RSB-

In

other

cases

HS

may

be

linked

to

a

systemic

condition

-LRB-

e.g.

cardiorespiratory

failure

-RRB-

or

cerebrovascular

disease

-LSB-

-RSB-

In

rare

cases

the

etiology

is

unknown

The

present

study

suggests

that

HSD

is

a

disorder

of

the

elderly

that

is

associated

with

more

marked

neuronal

and

synaptic

loss

and

greater

reactive

gliosis

than

that

seen

in

HS

associated

with

FTLD-U

This

argues

for

a

different

pathogenesis

B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hippocampal sclerosis dementia
immunohistochemistry
image analysis