Speech metrics, general disability, brain imaging and quality of life in multiple sclerosis.

BACKGROUND AND PURPOSE: Objective measurement of speech has shown promising results to monitor disease state in multiple sclerosis. In this study, we characterize the relationship between disease severity and speech metrics through perceptual (listener based) and objective acoustic analysis. We further look at deviations of acoustic metrics in people with no perceivable dysarthria. METHODS: Correlations and regression were calculated between speech measurements and disability scores, brain volume, lesion load and quality of life. Speech measurements were further compared between three subgroups of increasing overall neurological disability: mild (as rated by the Expanded Disability Status Scale ≤2.5), moderate (≥3 and ≤5.5) and severe (≥6). RESULTS: Clinical speech impairment occurred majorly in people with severe disability. An experimental acoustic composite score differentiated mild from moderate (P < 0.001) and moderate from severe subgroups (P = 0.003), and correlated with overall neurological disability (r = 0.6, P < 0.001), quality of life (r = 0.5, P < 0.001), white matter volume (r = 0.3, P = 0.007) and lesion load (r = 0.3, P = 0.008). Acoustic metrics also correlated with disability scores in people with no perceivable dysarthria. CONCLUSIONS: Acoustic analysis offers a valuable insight into the development of speech impairment in multiple sclerosis. These results highlight the potential of automated analysis of speech to assist in monitoring disease progression and treatment response.

Anti-inflammatory disease-modifying treatment and disability progression in primary progressive multiple sclerosis: a cohort study.

BACKGROUND AND PURPOSE: Treatment options in primary progressive multiple sclerosis (PPMS) are scarce and, with the exception of ocrelizumab, anti-inflammatory agents have failed to show efficacy in ameliorating disability progression. The aim of this study was to investigate a potential effect of anti-inflammatory disease-modifying treatment on disability outcomes in PPMS. METHODS: Using MSBase, a large, international, observational database, we identified patients with PPMS who were either never treated or treated with a disease-modifying agent. Propensity score matching was used to select subpopulations with similar baseline characteristics. Expanded Disability Status Scale (EDSS) outcomes were compared with an intention-to-treat and an as-treated approach in paired, pairwise-censored analyses. RESULTS: Of the 1284 included patients, 533 were matched (treated, n = 195; untreated n = 338). Median on-study pairwise-censored follow-up was 3.4 years (quartiles 1.2-5.5). No difference in the hazard of experiencing 3-month confirmed EDSS progression events was observed between the groups [hazard ratio (HR), 1.0; 95% confidence interval (CI), 0.6-1.7, P = 0.87]. We did not find significant differences in the hazards of confirmed EDSS improvement (HR, 1.0; 95% CI, 0.6-1.6, P = 0.91) or reaching a confirmed EDSS step ≥7 (HR, 1.1; 95% CI, 0.7-1.6, P = 0.69). CONCLUSION: Our pooled analysis of disease-modifying agents suggests that these therapies have no substantial effect on short- to medium-term disability outcomes in PPMS.

The occurrence of dystonia in upper-limb multiple sclerosis tremor.

BACKGROUND: The pathophysiology of multiple sclerosis (MS) tremor is uncertain with limited phenotypical studies available. OBJECTIVE: To investigate whether dystonia contributes to MS tremor and its severity. METHODS: MS patients (n = 54) with and without disabling uni- or bilateral upper limb tremor were recruited (39 limbs per group). We rated tremor severity, writing and Archimedes spiral drawing; cerebellar dysfunction (SARA score); the Global Dystonia Scale (GDS) for proximal and distal upper limbs, dystonic posturing, mirror movements, geste antagoniste, and writer's cramp. RESULTS: Geste antagoniste, mirror dystonia, and dystonic posturing were more frequent and severe (p < 0.001) and dystonia scores were correlated with tremor severity in tremor compared to non-tremor patients. A 1-unit increase in distal dystonia predicted a 0.52-Bain unit (95% confidence interval (CI) 0.08-0.97), p = 0.022) increase in tremor severity and a 1-unit (95% CI 0.48-1.6, p = 0.001) increase in drawing scores. A 1-unit increase in proximal dystonia predicted 0.93-Bain unit increase (95% CI 0.45-1.41, p < 0.001) in tremor severity and 1.5-units (95% CI 0.62-2.41, p = 0.002) increase in the drawing score. Cerebellar function in the tremor limb and tremor severity was correlated (p < 0.001). CONCLUSIONS: Upper limb dystonia is common in MS tremor suggesting that MS tremor pathophysiology involves cerebello-pallido-thalamo-cortical network dysfunction.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.

OBJECTIVES: We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). METHODS: Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. RESULTS: We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. CONCLUSIONS: A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS.

Onboarding of siponimod in secondary progressive multiple sclerosis patients in Australia: Novel, real-world evidence from the MSGo digital support programme.

Background: Siponimod is approved for use in people with secondary progressive multiple sclerosis (pwSPMS). An integrated digital platform, MSGo, was developed for pwSPMS and clinicians to help navigate the multiple steps of the pre-siponimod work-up. Objective: To explore real-world onboarding experiences of siponimod amongst pwSPMS in Australia. Methods: Retrospective, non-interventional, longitudinal, secondary analysis of data extracted from MSGo (20 April 2022). The primary endpoint was the average time for siponimod onboarding; secondary endpoints were adherence and sub-group analyses of variables influencing onboarding. Results: Mixed-cure modelling estimated that 58% of participants (N = 368, females 71%, median age of 59 years) registered in MSGo would ever initiate siponimod. The median time to initiation was 56 days (95% CI [47-59] days). Half of the participants cited 'waiting for vaccination' as the reason for initiation delay. Cox regression analyses found participants with a nominated care partner had faster onboarding (HR 2.1, 95% CI [1.5-3.0]) and were more likely to continue self-reporting daily siponimod dosing than were those without a care partner (HR 2.2, 95% CI [1.3-3.7]). Conclusions: Despite the limitations of self-reported data and the challenges of the COVID-19 pandemic, this study provides insights into siponimod onboarding in Australia and demonstrates the positive impact of care partner support.

Comparative effectiveness and cost-effectiveness of natalizumab and fingolimod in rapidly evolving severe relapsing-remitting multiple sclerosis in the United Kingdom.

AIM: To evaluate the real-world comparative effectiveness and the cost-effectiveness, from a UK National Health Service perspective, of natalizumab versus fingolimod in patients with rapidly evolving severe relapsing-remitting multiple sclerosis (RES-RRMS). METHODS: Real-world data from the MSBase Registry were obtained for patients with RES-RRMS who were previously either naive to disease-modifying therapies or had been treated with interferon-based therapies, glatiramer acetate, dimethyl fumarate, or teriflunomide (collectively known as BRACETD). Matched cohorts were selected by 3-way multinomial propensity score matching, and the annualized relapse rate (ARR) and 6-month-confirmed disability worsening (CDW6M) and improvement (CDI6M) were compared between treatment groups. Comparative effectiveness results were used in a cost-effectiveness model comparing natalizumab and fingolimod, using an established Markov structure over a lifetime horizon with health states based on the Expanded Disability Status Scale. Additional model data sources included the UK MS Survey 2015, published literature, and publicly available sources. RESULTS: In the comparative effectiveness analysis, we found a significantly lower ARR for patients starting natalizumab compared with fingolimod (rate ratio [RR] = 0.65; 95% confidence interval [CI], 0.57-0.73) or BRACETD (RR = 0.46; 95% CI, 0.42-0.53). Similarly, CDI6M was higher for patients starting natalizumab compared with fingolimod (hazard ratio [HR] = 1.25; 95% CI, 1.01-1.55) and BRACETD (HR = 1.46; 95% CI, 1.16-1.85). In patients starting fingolimod, we found a lower ARR (RR = 0.72; 95% CI, 0.65-0.80) compared with starting BRACETD, but no difference in CDI6M (HR = 1.17; 95% CI, 0.91-1.50). Differences in CDW6M were not found between the treatment groups. In the base-case cost-effectiveness analysis, natalizumab dominated fingolimod (0.302 higher quality-adjusted life-years [QALYs] and £17,141 lower predicted lifetime costs). Similar cost-effectiveness results were observed across sensitivity analyses. CONCLUSIONS: This MSBase Registry analysis suggests that natalizumab improves clinical outcomes when compared with fingolimod, which translates to higher QALYs and lower costs in UK patients with RES-RRMS.

There are several medications used to treat people with relapsing remitting multiple sclerosis, such as interferon-based therapies (Betaferon/Betaseron (US), Rebif, Avonex, Extavia), glatiramer acetate (Copaxone), teriflunomide (Aubagio), and dimethyl fumarate (Tecfidera), collectively named BRACETD. Other treatments for multiple sclerosis (MS) have a narrower use, such as natalizumab (Tysabri) or fingolimod (Gilenya), among others.This study objective was to assess how well natalizumab and fingolimod helped treating MS (clinical effectiveness) and subsequently estimate what the cost of these treatments is in comparison to the benefit they bring to people with rapidly evolving severe MS that use them in the United Kingdom (UK) (cost-effectiveness).We used an international disease registry (MSBase), which collects clinical data from people with MS in various centers around the world to compare the effectiveness of natalizumab, fingolimod and BRACETD treatments. We used a technique called propensity score matching to obtain results from comparable patient groups. People treated with natalizumab had better disease control, namely with fewer relapses and higher improvement on their disability level, than patients on fingolimod or BRACETD. Conversely, there were no differences between each group of people on a measure called disability worsening.Based on these clinical results, we built an economic model that simulates the lifetime costs and consequences of treating people with MS with natalizumab in comparison with fingolimod. We found that using natalizumab was less costly and was more effective compared to using fingolimod in UK patients.

Relationship between chronic demyelination of the optic nerve and short term axonal loss.

BACKGROUND: Axonal loss is a major determinant of disability in multiple sclerosis (MS). While acute inflammatory demyelination is a principal cause of axonal transection and subsequent axonal degeneration in acute disease, the nature of chronic axonal loss is less well understood. In the current study, the relationship between degree of chronic demyelination and axonal degeneration was investigated using optic neuritis (ON) as a model. METHOD: 25 patients with a first episode of unilateral ON, good recovery of visual function and concurrent brain or spinal cord MRI lesions were enrolled. Axonal loss was assessed using change in retinal nerve fibre layer (RNFL) thickness between 1 and 3 years after ON. Optic nerve conduction was evaluated using latency of multifocal visual evoked potentials (mfVEP). The level of mfVEP latency delay at 12 and 36 months was considered indicative of the degree of permanent demyelination. Data from 25 age and gender matched normal controls were used for comparison. RESULTS: RNFL thickness was significantly reduced in ON eyes at 12 months compared with controls but remained unchanged in fellow eyes. Average RNFL thickness demonstrated a small but significant reduction between 12 and 36 months for both ON and fellow eyes. Change in RNFL thickness between 12 and 36 months, however, did not correlate with the degree of mfVEP latency delay. CONCLUSION: The results, therefore, show no association between the degree of permanent optic nerve demyelination (as measured by latency delay) and progressive axonal degeneration, at least in the early stages of the disease. The fact that fellow eyes demonstrated a similar degree of progressive axonal loss supports this suggestion.

Lesion Volume in Relapsing Multiple Sclerosis is Associated with Perivascular Space Enlargement at the Level of the Basal Ganglia.

BACKGROUND AND PURPOSE: Perivascular spaces surround the blood vessels of the brain and are involved in neuroimmune functions and clearance of metabolites via the glymphatic system of the brain. Enlarged perivascular spaces could be a marker of dysfunction in these processes and, therefore, are highly relevant to monitoring disease activity in MS. This study aimed to compare the number of enlarged perivascular spaces in people with relapsing MS with MR imaging markers of inflammation and brain atrophy. MATERIALS AND METHODS: Fifty-nine patients (18 with clinically isolated syndrome, 22 with early and 19 with late relapsing-remitting MS) were scanned longitudinally (mean follow-up duration = 19.6 [SD, 0.5] months) using T2-weighted, T1-weighted, and FLAIR MR imaging. Two expert raters identified and counted enlarged perivascular spaces on T2-weighted MR images from 3 ROIs (the centrum semiovale, basal ganglia, and midbrain). Baseline and change with time in the number of enlarged perivascular spaces were correlated with demographics and lesion and brain volumes. RESULTS: Late relapsing-remitting MS had a greater average number of enlarged perivascular spaces at baseline at the level of the basal ganglia (72.3) compared with early relapsing-remitting MS (60.5) and clinically isolated syndrome (54.7) (F = 3.4, P = .042), and this finding correlated with lesion volume (R = 0.44, P = .0004) but not brain atrophy (R = -0.16). Enlarged perivascular spaces increased in number with time in all regions, and the rate of increase did not differ among clinical groups. CONCLUSIONS: Enlarged perivascular spaces at the level of the basal ganglia are associated with greater neuroinflammatory burden, and the rate of enlargement appears constant in patients with relapsing-remitting disease phenotypes.

Canine hip extension range during gait.

Assessment of canine gait is frequently used by veterinary clinicians to establish the presence of orthopaedic pain. As up to 30% of canine orthopaedic conditions affect the pelvic limb, knowledge of pelvic limb biomechanics during gait is very important. Previous studies have investigated the biomechanics at the tarsus and stifle, but little information is available regarding hip motion during gait. The aim of this study was to determine the maximum hip extension range achieved during the stance phase of gait in normal canines. In addition, this study aimed to determine the difference between maximum passive hip extension and maximum hip extension during gait. Using a sample of 30 morphologically similar normal dogs, mean maximum passive hip extension was measured using a goniometer and mean maximum hip extension range during gait was determined videographically. Inter- and intra-assessor reliability studies performed at the start of the study showed that the measurement tools and techniques used in this study were valid and reliable. The goniometric data showed that mean maximum passive hip extension range was 162.44 degrees (+/-3.94) with no significant difference between the left and the right hind limbs. The videographic data showed that mean maximum hip extension range during gait was 119.9 degrees (+/-9.26) with no significant difference between the left and right hind limbs. The results of this study provided reference values for active and passive hip extension range and showed that the degree of hip extension range required for normal gait is significantly less than maximum passive hip extension range.

Monitoring cognitive change in multiple sclerosis using a computerized cognitive battery.

BACKGROUND: Cognitive monitoring that can detect short-term change in multiple sclerosis is challenging. Computerized cognitive batteries such as the CogState Brief Battery can rapidly assess commonly affected cognitive domains. OBJECTIVES: The purpose of this study was to establish the acceptability and sensitivity of the CogState Brief Battery in multiple sclerosis patients compared to controls. We compared the sensitivity of the CogState Brief Battery to that of the Paced Auditory Serial Addition Test over 12 months. METHODS: Demographics, Expanded Disability Status Scale scores, depression and anxiety scores were compared with CogState Brief Battery and Paced Auditory Serial Addition Test performances of 51 patients with relapsing-remitting multiple sclerosis, 19 with secondary progressive multiple sclerosis and 40 healthy controls. Longitudinal data in 37 relapsing-remitting multiple sclerosis patients were evaluated using linear mixed models. RESULTS: Both the CogState Brief Battery and the Paced Auditory Serial Addition Test discriminated between multiple sclerosis and healthy controls at baseline (p<0.001). CogState Brief Battery tasks were more acceptable and caused less anxiety than the Paced Auditory Serial Addition Test (p<0.001). In relapsing-remitting multiple sclerosis patients, reaction time slowed over 12 months (p<0.001) for the CogState Brief Battery Detection (mean change -34.23 ms) and Identification (-25.31 ms) tasks. Paced Auditory Serial Addition Test scores did not change over this time. CONCLUSIONS: The CogState Brief Battery is highly acceptable and better able to detect cognitive change than the Paced Auditory Serial Addition Test. The CogState Brief Battery could potentially be used as a practical cognitive monitoring tool in the multiple sclerosis clinic setting.

Myotonia dystrophica: Studies on gonadal function using luteinizing hormone-releasing hormone (LRH).

Eight men and four women with myotonia dystrophica were investigated. The gonadotropin responses to 100 mug luteinizing hormone-releasing hormone (LRH) were studied, and serum testosterone assayed. In the men with testicular atrophy a high basal follicle stimulating hormone was found with an exaggerated response to LRH. Although the mean basal luteinizing hormone was not significantly elevated, there was an excessive response to LRH, which was possibly conditioned by the lower mean testoerone levels in this group. This study provides biochemical indices of both the primary nature of the testicular damage and the disproportionately greater involvement of the seminiferous tubules. The pituitary-gonadal axis was normal in the women.

Human chorionic gonadotropin in the ectopic gestation.

The ectopic pregnancy is a relatively common condition in the south African black patients. The beta-specific subunit radioimmunoassay for human chorionic gonadotrophin (HCG) was utilized in procuring information in our series of 30 patients. Levels of the hormone were significantly lower when compared to normal gestation of similar duration, never exceeding 2000 mIU/ml. The clearance rate of HCG following normal vaginal delivery was about 24 h and less variable than that of ectopic gestation. The half-life clearance rate of HCG in the ectopics could be divided into three phases, suggestive of HCG compartmentalization. The possible buffering effect of this hormone in the maintenance of the receptor-saturated pregnancy is discussed. The possibility that the HCG produced by the normal pregnancy is dissimilar to that of ectopic is speculated upon.

Attitudes of diploma-prepared and graduate registered nurses. Towards nursing models: a comparative study.

UNLABELLED: Currently, nursing theory is not a South African Nursing Council requirement for pre-registration nursing education curricula. AIM OF THE STUDY: to determine registered nurses' (RNs) attitudes towards nursing models. METHODOLOGY: replication of McKenna's (1994) 20-item Likert-type self-completion questionnaire with adaptations. RESEARCH SETTING: A 1,600-bedded State-funded academic hospital in Cape Town, South Africa. STUDY SUBJECTS: Two groups: RNs who had nursing degrees and RNs who had a traditional diploma training. FINDINGS: Results from the Mann-Whitney U-test for independent samples indicates that at the 0.05 level of significance there was no difference in how each group of RNs responded to 19 statement items so there was not enough evidence to reject the Null Hypothesis for these statements (P < 0.05). However, as a group, the diploma-prepared RNs appeared to have a more positive attitude than the graduate RNs towards nursing models.

MicroRNAs in fruit trees: discovery, diversity and future research directions.

Since the first description of microRNAs (miRNAs) 20 years ago, the number of miRNAs identified in different eukaryotic organisms has exploded, largely due to the recent advances in DNA sequencing technologies. Functional studies, mostly from model species, have revealed that miRNAs are major post-transcriptional regulators of gene expression in eukaryotes. In plants, they are implicated in fundamental biological processes, from plant development and morphogenesis, to regulation of plant pathogen and abiotic stress responses. Although a substantial number of miRNAs have been identified in fruit trees to date, their functions remain largely uncharacterised. The present review aims to summarise the progress made in miRNA research in fruit trees, focusing specifically on the economically important species Prunus persica, Malus domestica, Citrus spp, and Vitis vinifera. We also discuss future miRNA research prospects in these plants and highlight potential applications of miRNAs in the on-going improvement of fruit trees.

Apolipoprotein genotype does not influence MS severity, cognition, or brain atrophy.

BACKGROUND: The influence of APOE allelic heterogeneity on multiple sclerosis (MS) disease severity has been reported in multiple datasets with conflicting results. Several studies have reported an unfavorable association of APOE epsilon4 with more severe clinical disease course while, in contrast, APOE epsilon2 has been associated with a more benign disease course. In this study, we examine the influence of heterogeneity of the APOE gene on disease severity in a large, Australian, population-based MS cohort. METHODS: Associations between APOE allele status, 2 promoter region single nucleotide polymorphisms (-219 G/T and +113 C/G), and 4 measures of disease severity were tested in 1,006 patients with relapsing-remitting MS and secondary progressive MS: 1) Multiple Sclerosis Severity Score; 2) Progression Index (Expanded Disability Status Scale/disease duration); 3) age at first symptom; and 4) interval between the first and second attack. The Symbol Digit Modalities Test was used as a single cognitive marker in 889 patients. Brain atrophy was measured in 792 patients using the intercaudate ratio. APOE epsilon4 and epsilon3 carriers were stratified by -219 G/T or +113 C/G to investigate haplotypic heterogeneity in the APOE gene region. RESULTS: In this MS study, neither APOE allele status nor promoter region heterogeneity at positions -219 G/T or +113 C/G influenced the clinical disease severity, cognition, or cerebral atrophy. CONCLUSIONS: Allelic and haplotypic heterogeneity of the APOE gene region does not influence multiple sclerosis disease course in this well-defined Australian multiple sclerosis cohort.

Viable pregnancy after prolonged periods of secondary infertility.

Case records of 206 pregnant patients who had not had a viable pregnancy for 10 years or more, are presented. In the antenatal period, 59.7% of patients went through their pregnancy with no complications. The commonest complications were pre-eclampsia and non-proteinuric hypertension. Labour was essentially normal in the majority of cases, and of the 183 patients who were allowed to labour, 171 (93.4%) were delivered vaginally. Caesarean section was performed in 17% of cases, which is 3 times the over-all Caesarean section rate for the hospital during the same period. The indications for Caesarean section are appreciably less restricted in these patients. There was a two-fold increase in the incidence of breech presentation. This may be partly explained by the higher incidence of premature births found in this group of patients. The over-all risk to the infant is increased, mainly because of prematurity, but also owing to a slight increase in the number of congenital abnormalities.

Inclusion of xylan in a medium for the enumeration of total culturable rumen bacteria.

The influence of the inclusion of xylan in a medium for enumeration of total culturable rumen bacteria was investigated. Maximum colony numbers were obtained on a medium, GCSX-2, which contained 0.033% each glucose and cellobiose and 0.067% each soluble starch and xylan. This medium gave higher colony counts than either medium 98-5 of Bryant and Robinson (J. Dairy Sci. 44:1446-1456, 1961), medium 98-5 of Chung and Hungate (Appl. Environ. Microbiol. 32:649-652, 1976), containing an added lucerne (hemicellulose + cellulose) fiber substrate, or medium GCSX-2 with the added lucerne (hemicellulose + cellulose) fraction. The time of collection of rumen fluid influenced the colony counts on the media containing the lucerne fiber substrate but was without effect on medium GCSX-2.

Suppression of postpartum lactation with furosemide.

A regimen of fursemide and moderate restriction of fluid intake was followed in 120 postpartum women to suppress lactation. The methods and results are presented, and possible mechanisms whereby furosemide may suppress lactation are discussed.

Diamine extension of glutaraldehyde crosslinks mitigates bioprosthetic aortic wall calcification in the sheep model.

We previously have been able to show that fixation at increasing concentrations of glutaraldehyde (GA) leads to mitigated rather than facilitated tissue calcification. The purpose of the present study was to introduce additional crosslinks and provide evidence that crosslink density may be an underlying inhibitory principle. Entire aortic roots were chosen to verify the concept on the challenging aortic wall tissue. Porcine aortic roots were crosslinked with 0.2% GA, 3%GA, and 3% GA containing an interim step that introduced diamine bridges. Crosslink efficiency was determined on the basis of shrinkage temperature (SrT degrees ), resistance to protease digestion (RPD), residual amine analysis (RA), and tensile modulus (E(10)). Calcium levels, calcification patterns, and inflammation were assessed after 6 and 24 weeks of implantation in a sheep circulatory model. Crosslink efficiency in aortic wall tissue was moderately affected by increasing the fixative concentration from 0.2% GA to 3% GA (SrT degrees from 85.7 degrees +/- 0.3 degrees to 87.5 degrees +/- 0.3 degrees C, p < 0.002; RPD from 24.2 +/- 1.2 to 29.1 +/- 0.7%, p < 0.003; RA from 0.069 +/- 0.004 to 0.058 +/- 0.003 micromol/mg, p < 0.03, and E(10) from 1.9 +/- 0.11 to 2.94 +/- 0.34 MPa, p < 0.01), but it was distinctly enhanced when diamine bridges were introduced (SrT degrees from 87.5 degrees +/- 0.3 degrees to 93.4 degrees +/- 0.3 degrees C, p << 0.0001; RPD from 29.1 +/- 0.7 to 68.4 +/- 1.8%, p << 0.0001; and E(10) from 2.94 +/- 0.34 to 6.80 +/- 0.61 MPa, p < 0.0003). Aortic wall calcification was reduced significantly by increasing the GA concentration from 0.2 to 3% [37.8%, p = 0.076 (6 weeks) and 34.0%, p = 0.008 (24 weeks)] and further reduced by the introduction of additional diamine [84.0%, p = 0.006 (6 weeks) and 29.8%, p = 0.037 (24 weeks)]. The combined effect of increased GA concentration plus an interim diamine step on aortic wall tissue resulted in a 90% and 53.7% reduction of calcification after 6 weeks and 24 weeks, respectively. The correlation coefficients between calcification and SrT degrees, RDP, and E(10) was -0.9767, -0.9460, and -0.9740, respectively (6 weeks). The inflammatory host reaction regularly found in 0.2% fixed tissue was practically abolished through the introduction of diamine bridges. Our study demonstrated a distinct correlation between the mitigation of aortic wall calcification and three parameters used to assess crosslink density.